Pharmaceutical preparations of glutathione and methods of administration thereof

ABSTRACT

A method of altering an expression of a gene product in cells or an organism, comprising orally administering glutathione in an effective amount and under such conditions to alter a redox potential in the cells. The gene expression may be sensitive to redox potential through one or more of a process of induction, transcription, translation, post-translational modification, release, and/or through a receptor mediated process. The glutathione is preferably administered as an oral bolus of encapsulated pharmaceutically stabilized glutathione in a rapidly dissolving formulation to a mammal on an empty stomach.

This application claims benefit to U.S. provisional 60/034,101 filedDec. 31, 1996 which is a con of Ser. No. 09/002,100 filed Dec. 31, 1997now abandoned which is a 371 of PCT/US97/238,790 filed Dec. 31, 1997which is a con of Ser. No. 09/331,947 filed Jun. 28, 1999.

FIELD OF THE INVENTION

The present invention relates to the field of reduction/oxidation(redox) potential altering pharmaceutical preparations, and methods foradministration thereof, and more particularly to the use of theantioxidant agent glutathione as a cellular redox altering therapy.

BACKGROUND OF THE INVENTION

The ubiquitous tripeptide L-glutathione (GSH)(gamma-glutamyl-cysteinyl-glycine), is a well known biologicalantioxidant, and in fact is believed to be the primary intracellularantioxidant for higher organisms. When oxidized, it forms a dimer(GSSG), which may be recycled in organs having glutathione reductase.Glutathione may be transported through membranes by the sodium-dependentglutamate pump. Tanuguchi, N., et al. Eds., Glutathione Centennial,Academic Press, New York (1989), expressly incorporated herein byreference.

GSH is known to function directly or indirectly in many importantbiological phenomena, including the synthesis of proteins and DNA,transport, enzyme activity, metabolism, and protection of cells fromfree-radical mediated damage. GSH is one of the primary cellularantioxidants responsible for maintaining the proper oxidation statewithin the body. GSH is synthesized by most cells, and is also suppliedin the diet. GSH has been shown to recycle oxidized biomolecules back totheir active, reduced forms.

Because of the existing mechanisms for controlling interconversion ofreduced and oxidized glutathione, an alteration of the level of reducedglutathione (GSH), e.g., by administration of GSH to an organism willtend to shift the cells of the organism to a more reduced redoxpotential. Likewise, subjecting the organism to oxidative stress or freeradicals will tend to shift the cells to a more oxidized potential. Itis well known that certain cellular processes are responsive to redoxpotential.

Reduced glutathione (GSH) is, in the human adult, produced from oxidizedglutathione (GSSG) primarily by the liver, and to a smaller extent, bythe skeletal muscle, red blood cells, and white cells. About 80% of the8-10 grams glutathione produced daily is produced by the liver anddistributed through the blood stream to the other tissues.

A deficiency of glutathione in cells may lead to excess free radicals,which cause macromolecular breakdown, lipid peroxidation, buildup oftoxins, and ultimately cell death. Because of the importance ofglutathione in preventing this cellular oxidation, glutathione iscontinuously supplied to the tissues. However, under certain conditions,the normal, physiologic supplies of glutathione are insufficient,distribution inadequate or local oxidative demands too high to preventcellular oxidation. Under certain conditions, the production of anddemand for glutathione are mismatched, leading to insufficient levels onan organismal level. In other cases, certain tissues or biologicalprocesses consume glutathione so that the intracellular levels aresuppressed. In either case, by increasing the serum levels ofglutathione, increased amounts may be directed into the cells. Infacilitated transport systems for cellular uptake, the concentrationgradient which drives uptake is increased.

As with all nutrients, eating or orally ingesting the nutrient wouldgenerally be considered a desired method for increased body levelsthereof. Thus, attempts at oral glutathione treatments were known, andindeed the present inventors hereof previously suggested oralglutathione administration for various indications. The protocols foradministration of glutathione, however, were not optimized and thereforethe bioavailability of the glutathione was unassured and variable. Priorpharmaceutical attempts by others to safely, effectively and predictablyraise intracellular GSH through oral therapy with GSH have not met withdemonstrated success. Experts generally believe that beneficialphysiological effects of orally administered glutathione are difficultor impossible to achieve, or the efficiency is so low as to makesupplementation by this route unproductive.

Because of the poor or variable results obtained, the art generallyteaches that oral administration of glutathione is ineffective, forcingadministration or supplementation by other routes, principallyintravenously, but also by alveolar inhalation. Orally absorbed prodrugsand precursors have also been proposed or used. A known pharmacologicalregimen provides intravenous glutathione in combination with anotheragent, such as cis-platinum (a free radical associated metal drug),doxorubicin, or daunorubicin (free radical associated drugs whichinteract with nucleic acid metabolism), which produced toxic sideeffects related to free radical reactions.

The ability to harness GSH, which is a powerful, but safe substance,into an effective oral pharmaceutical had not been accomplished in thepast, because of molecular instability, poor gastrointestinal absorptionthrough existing protocols and resulting inability to reliably effectincreases in intracellular GSH levels. Administering sufficient amountsto achieve physiological benefit using known oral administrationprotocols might lead to cysteine related kidney stones, gastric distressor flatulence.

Glutathione is relatively unstable in alkaline or oxidativeenvironments, and is not absorbed by the stomach. It is believed thatglutathione is absorbed, after oral administration, if at all, in thelatter half of the duodenum and the beginning of the jejunum. It wasalso believed that orally administered glutathione would tend to bedegraded in the stomach, and that it is particularly degraded underalkaline conditions by desulfurases and peptidases present in theduodenum. Thus, known protocols for oral administration of glutathioneinvolved administered with meals or after eating to buffer pH extremesand dilute degradative enzymes. This protocol, however, has the effectof diluting the glutathione and delaying absorption. Studies directed atdetermining the oral bioavailability of glutathione under suchcircumstances showed poor absorption, and therefore such administrationwas seen as of little benefit.

Therefore, while oral dosage forms of glutathione were known, theclinical benefits of these formulations were unproved and, given thelack of predictability of their effect, these formulations were not usedfor the treatment of specific conditions, nor proven to have effect.Further, the known protocols for administration of glutathione did notprovide convenience and high bioavailability.

The prior art thus suggests that glutathione esters might be suitable asorally bioavailable sources of glutathione, which are stable and may berapidly absorbed. However, these are both more expensive thanglutathione itself and have proven toxic.

Pure glutathione forms a flaky powder that retains a static electricalcharge, due to triboelectric effects, making processing and formulationdifficult. The powder particles may also have an electrostaticpolarization, which is akin to an electret. Glutathione is a strongreducing agent, so that autooxidation occurs in the presence of oxygenor other oxidizing agents. U.S. Pat. No. 5,204,114, Demopoulos et al.,expressly incorporated herein by reference in its entirety, provides amethod of manufacturing glutathione tablets and capsules by the use ofcrystalline ascorbic acid as an additive to reduce triboelectric effectswhich interfere with high speed equipment and maintaining glutathione ina reduced state. A certain crystalline ascorbic acid is, in turn,disclosed in U.S. Pat. No. 4,454,125, Demopoulos, expressly incorporatedby reference herein in its entirety. This crystalline form is useful asa lubricating agent for machinery. Ascorbic acid has the advantage thatit is well tolerated, antioxidant, and reduces the net static charge onthe glutathione.

In synthesizing glutathione in the body, cysteine, a thiol amino acid isrequired. Since the prior art suggests that oral administration ofglutathione itself would be ineffective, prodrugs or precursor therapywas advocated. Therefore, the prior art suggests administration ofcysteine, or a more bioavailable precursor of cysteine, N-acetylcysteine (NAC). While cysteine and NAC are both, themselves,antioxidants, their presence competes with glutathione for resources incertain reducing (GSH recycling) pathways. Since glutathione is aspecific substrate for many reducing pathways, the loading of a hostwith cysteine or NAC may result in less efficient utilization orrecycling of glutathione. Thus, cysteine and NAC are not ideal GSHprodrugs. NAC has also demonstrated some neurotoxicity. Thus, while GSHmay be degraded, transported as amino acids, and resynthesized in thecell, there may also be circumstances where GSH is transported intocells without degradation; and in fact the administration of cysteine orcysteine precursors may interfere with this process.

A number of disease states have been specifically associated withreductions in glutathione levels. Depressed glutathione levels, eitherlocally in particular organs, or systemically, have been associated witha number of clinically defined diseases and disease states. Theseinclude HIV/AIDS, diabetes and macular degeneration, all of whichprogress because of excessive free radical reactions and insufficientGSH. Other chronic conditions may also be associated with GSHdeficiency, including heart failure and coronary artery restenosis postangioplasty.

For example, diabetes afflicts 8% of the United States population andconsumes nearly 15% of all United States healthcare costs. HIV/AIDS hasinfected nearly 1 million Americans. Current therapies cost in excess of$20,000 per year per patient, and are rejected by, or fail in 25% to 40%of all patients. Macular degeneration presently is considered incurable,and will afflict 15 million Americans by 2002.

Clinical and pre-clinical studies have demonstrated the linkage betweena range of free radical disorders and insufficient GSH levels. Newlypublished data implies that diabetic complications are the result ofhyperglycemic episodes that promote glycation of cellular enzymes andthereby inactivate GSH synthetic pathways. The result is GSH deficiencyin diabetics, which may explain the prevalence of cataracts,hypertension, occlusive atherosclerosis, and susceptibility toinfections in these patients.

GSH functions as a detoxicant by forming GSH S-conjugates withcarcinogenic electrophiles, preventing reaction with DNA, and chelationcomplexes with heavy metals such as nickel, lead, cadmium, mercury,vanadium, and manganese. GSH also plays a role in metabolism of variousdrugs, such as opiates. It has been used as an adjunct therapy totreatment with nephrotoxic chemotherapeutic agents such as cisplatin,and has been reported to prevent doxorubicin-induced cardiomyopathy. GSHis also an important factor in the detoxification of acetaminophen andethanol, two powerful hepatotoxins. See:

Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on thedecomposition of glutathione. I. Decomposition in solid state. Chem.Pharm. Bull. 26: 2081-91, 1978.

Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition ofglutathione. II. Anaerobic decomposition in aqueous solution. Chem.Pharm. Bull. 28: 514-20, 1980.

Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition ofglutathione. III. Peptide bond cleavage and desulfurization in aqueoussolution. Chem. Pharm. Bull. 28: 521-28, 1980.

Hagen, T. M., Aw, T. Y., and Jones, D. P.: Glutathione uptake andprotection against oxidative injury in isolated kidney cells. KidneyIntl. 34: 74-81, 1988.

Lash, L. H., and Jones, D. P.: Distribution of oxidized and reducedforms of glutathione and cysteine in rat plasma. Arch. Biochem. Biophys.240: 583-92, 1985.

Meister, A.: Selective modification of glutathione metabolism. Science220: 472-477, 1983.

Meister, A. and Anderson, M. E.: Glutathione. Ann. Rev. Biochem. 52:711-60, 1983.

Riley, R. J., Spielberg, S. P., Leeder, J. S.: A comparative study ofthe toxicity of chemically reactive xenobiotics towards adherent cellcultures: selective attenuation of menadione toxicity by buthioninesulphoximine pretreatment. J. Pharmacol. 45 (4): 263-267, 1993.

Wierzbicka, G. T., Hagen, T. M. & Jones, D. P.: Glutathione in food. J.Food Comp. Anal. 2: 327-337, 1989.

Bravenboer, B., Kappelle, A. C., Hamers, F. P., van Buren, T., Erkelens,D. W. & Gispen, W. H.: Potential use of glutathione for the preventionand treatment of diabetic neuropathy in the streptozocin-induceddiabetic rat. Diabetologia 35: 813-817, 1992.

Cavaletti, E., Tofanetti, O. & Zunino F.: Comparison of reducedglutathione with 2-mercaptoethane sulfonate to preventcyclophosphamide-induced urotoxicity. Cancer Letters 32: 1, 1986.

Hamers, F. P., Brakkee, J. H., Cavalletti, E., Tedeschi, M., Marmonti,L., Pezzoni, G., Neijt, J. P. & Gispen, W. H.: Reduced glutathioneprotects against cisplatin-induced neurotoxicity in rats. Cancer Res.53: 544-549, 1993.

Kromidas, L., Trombetta, L. D., and Jamall, I. S.: The protectiveeffects of glulathione against methylmercury cytotoxicity. Toxicol.Letters 51: 67-80, 1990.

Novi, A. M., Flohe, R., and Stukenkemper, S.: Glutathione and aflatoxinB1-induced liver tumors: requirement for an intact glutathione moleculefor regression of malignancy in neoplastic tissue. Ann. NY Acad. Sci.397: 62-71, 1982.

Rao, R. D. N., Fischer, V., and Mason, R. P.: Glutathione and ascorbatereduction of the acetaminophen radical formed by peroxidase. J. Biol.Chem. 265: 844-7, 1990.

Skoulis, N. P., James, R. C., Harbison, R. D. and Roberts, S. M.:Depression of hepatic glutathione by opioid analgesic drugs in mice.Toxicol. Appl. Pharmacol. 99: 139-47, 1989.

Villani, F., Galimberti, M., Zunino, F., Monti, E., Rozza, A., Favalli,L. & Poggi, P.: Prevention of doxorubicin-induced cardlomyopathy byreduced glutathione. Cancer Chemother. Pharmacol. 28: 365-369, 1991.

Wagner, G., Frenzel, H., Wefers, H. and Sies, H.: Lack of effect oflong-term glutathione administration on aflatoxin B1-induced hepatoma inmale rats. Chem. Biol. Interactions 53: 57-68, 1985.

Yoda, Y., Nakazawa, M., Abe, T. & Kawakami, Z.: Prevention ofDoxorubicin myocardial toxicity in mice by reduced glutathione. CancerResearch 46: 2551, 1986.

Younes, M., and Strubelt, O.: Protection by exogenous glutathioneagainst hypoxic and cyanide-induced damage to isolated perfused ratlivers. Toxicol. Letters 50: 229-236, 1990.

McCartney, M. A.: Effect of glutathione depletion on morphine toxicityin mice. Biochem. Pharmacol. 38: 207-9, 1989.

Ishida, T., Kumagai, Y., Ikeda, Y., Ito, K., Yano, M., Toki, S.,Mihashi, K., Fujioka, T., Iwase, Y. and Hachiyama, S.:(8S)-(glutathion-S-YL)dihydromorphinone, a novel metabolite kof morphinefrom guinea pig bile. Drug. Metab. Dispos. 17: 77-81, 1989.

Nagamatsu, K., Kido, Y., Teroa, T, Ishida, T. and Toki, S.: Protectiveeffect of sulfhydryl compounds on acute toxicity of morphinone. LifeSci. 30: 1121-27, 1982.

(1) HIV

High GSH levels have been demonstrated to be necessary for properfunctioning of platelets, vascular endothelial cells, macrophages,cytotoxic T-lymphocytes, and other immune system components. Recently ithas been discovered that HIV-infected patients exhibit low GSH levels inplasma, in other fluids, and in certain cell types like macrophages,which does not appear to be due to defects in GSH synthesis.

Dröge, W., Pottmeyer-Gerber, C., Schmidt, H. & Nick, S.: Glutathioneaugments the activation of cytotoxic T lymphocytes in vivo. Immunobiol.172: 151-156, 1986.

Dröge, W., Eck, H. P., Gmunder, H., and Mihm, S.: Modulation oflymphocyte functions and immune responses by cysteine and cysteinederivatives. Amer. J. Medicine 91 (3C): 140S-144S, 1991.

Furukawa, T., Meydani, S. N. & Blumberg, J. B.: Reversal ofage-associated decline in immune responsiveness by dietary glutathionesupplementation in mice. Mech. Ageing Dev. 38: 107-117, 1987.

Franklin, R. A., Yong, M. L., Arkins, S., and Kelley, K. W.: Glutathioneaugments in vitro proliferative responses of lymphocytes to concanavalinA to a greater degree in old than in young rats. J. Nutr. 120: 1710-17,1990.

Kavanaugh, T. J., Grossman, A., Jaecks, E. P, Jinneman, J. C., Eaton, D.L., Masrtin, G. M., and Rabinovitch, P. S.: Proliferative capacity ofhuman peripheral lymphocytes sorted on the basis of glutathione content.J. Cell. Physiol. 145: 472-80, 1990.

Robinson, M. K, Rodrick, M. L., Jacobs, D. O., Rounds, J. D., Collins,K. H., Saproschetz, I. B., Mannick, J. A., and Wilmore, D. W.:Glutathione depletion in rats impairs T-cell and macrophage immunefunction. Arch. Surg. 128: 29-35, 1993.

Suthanthiran, M., Anderson, M. E., Sharma, V. K. & Meister, A.:Glutathione regulates activation-dependent DNA synthesis in highlypurified normal human T lymphocytes stimulated via the CD2 and CD3antigens. Proc. Natl. Acad. Sci. USA 87: 3343-3347, 1990.

GSH has been shown to inhibit HIV replication in chronically-infectedcells and in cells acutely infected in vitro. This makes GSH replacementtherapy attractive, because it has the potential to interfere with theexpression of the integrated HIV genome, a site that is not attacked bythe currently employed antiretrovirals (AZT, ddI, ddC, D4T). GSH mayalso have benefits in countering the excess free radical reactions inHIV infection, which may be attributable to: 1) the hypersecretion ofTNF-α by B-lymphocytes, in HIV infection, and 2) the catalysis ofarachidonic acid metabolism by the GP-120 protein of HIV. Thephysiologic requirements for GSH by key cell types of the immune system,and the ability of macrophages to take up intercellular GSH, as well asto metabolically interact with T-lymphocytes to indirectly cause theirGSH to increase, offer additional reasons to attempt to correct the GSHdeficiency in HIV/AIDS.

In other new data dealing with HIV infections, the March 1997 issue ofthe Proceedings of the National Academy of Sciences (PNAS) established “. . . GSH deficiency as a key determinant of survival in HIV disease . .. ” GSH deficiency is associated with impaired survival in HIV disease(PNAS. Vol. 94, pp. 1967-1972). The quest to raise GSH levels in cellsis widely recognized as being extremely important in HIV/AIDS and otherdisorders, because the low cellular GSH levels in these diseaseprocesses permit more and more free radical reactions to propel thedisorders.

HIV is known to start pathologic free radical reactions that lead to thedestruction of GSH, as well as exhaustion of other antioxidant systemsand destruction of cellular organelles and macromolecules. Inpre-clinical studies, GSH stops the replication of the virus at a uniquepoint, and specifically prevents the production of toxic free radicals,prostaglandins, TNF-α, interleukins, and a spectrum of oxidized lipidsand proteins that are immunosuppressive, cause muscle wasting andneurologic symptoms. Restoring GSH levels could slow or stop thediseases progression, safely and economically.

In mammalian cells, oxidative stresses, i.e., low intracellular levelsof reduced GSH, and relatively high levels of free radicals, activatecertain cytokines, including NF-κB and TNF-α, which, in turn, activatecellular transcription of the DNA to mRNA, resulting in translation ofthe mRNA To A Polypeptide Sequence. See, Sonia Schoonbroodt, SylvieLegrand-Poels, Martin Best-Belpomme and Jacques Piette; Activation ofthe NF-κB transcription factor in a T-lymphocytic cell line byhypochlorous acid, Biochem. J. (1997) 321, 777-785, Flohé, L.,Brigelius-Flohé, R., Saliou, C., Traber, M. G. and Packer, L., Redoxregulation of NF-kappa B activation. (1997) Free Radical Biology andMedicine, 22: 1115-1126. Antioxidants have been shown to block theinduction of NF-κB by oxidant agents. In a virus-infected cell, theviral genome is transcribed, resulting in viral RNA production,generally necessary for viral replication of RNA viruses andretroviruses. These processes require a relatively oxidized state of thecell, a condition which results from stress, low glutathione levels, orthe production of reduced cellular products. The mechanism thatactivates cellular transcription is evolutionarily highly conserved, andtherefore it is unlikely that a set of mutations would escape thisprocess, or that an organism in which mutated enzyme and receptor geneproducts in this pathway would be well adapted for survival. Thus, bymaintaining a relatively reduced state of the cell (relatively reducedredox potential), viral transcription, a necessary step in late stageviral replication, is impeded.

The amplification effect of oxidative intracellular conditions on viralreplication is compounded by the actions of various viruses and viralproducts that degrade GSH. For example, GP-120, an HIV surfaceglycoprotein having a large number of disulfide bonds, and normallypresent on the surface of infected cells, oxidizes GSH, resulting inreduced intracellular GSH levels. On the other hand, GSH reducesdisulfide bonds of GP-120, decreasing or eliminating its biologicalactivity, which in turn is necessary for viral infectivity. GSHtherefore interferes with the production of such oxidized proteins, anddegrades them once formed. GSH also participates in the destruction ofhydrogen peroxide, which is a long-lived oxidative messenger which hasbeen implicated in activating NF-κB. R. Schreck, P. Rieber & P. A.Baeuerle; Reactive oxygen intermediates as apparently widely usedmessengers in the activation of the NF-kappa B transcription factor andHIV-1, EMBO J 10: 2247-2258 (1991).

In a cell which is actively replicating viral gene products, a cascadeof events may occur which allow the cell to pass from a relativelyquiescent stage with low viral activity to an active stage with massiveviral replication and cell death, accompanied by a change in cellularredox potential; by maintaining adequate GSH levels, this cascade may beimpeded.

Thus, certain viral infections, such as HIV, are associated with reducedGSH levels, and it is believed that by increasing intracellular GSHlevels in infected cells, as well as increasing extracellular GSH, thereplication of HIV may be interfered with, and the cascade of eventsdelayed or halted. It is noted that AIDS may also be associated withreduced GSSG levels, implying an interference with de novo synthesis ofGSH as well as the oxidation of existing GSH discussed above.

Initially after infection with HIV, there is an intense viral infectionsimulating a severe case of the flu, with massive replication of thevirus. This acute phase passes within weeks, spontaneously, as the bodymounts a largely successful immune defense. Thereafter, the individualhas no outward manifestations of the infection. However, the viruscontinues to replicate, insidiously, within immune system tissues andcells, like lymph nodes, lymphoid nodules and special multidendriticcells that are found in various body cavities.

This infection is not just a viral problem. The virus, in addition toreplicating, causes excessive production of various free radicals andvarious cytokines in toxic or elevated levels. The latter are normallyoccurring biochemical substances that signal numerous reactions, usuallyexisting in minuscule concentrations. Eventually, after an average of7-10 years of seemingly quiescent HIV infection, the corrosive freeradicals and the toxic levels of cytokines begin to cause symptoms, andfailures in the immune system begin. Toxic factors, such as 15-HPETE,which is immunosuppressive, and TNF-α, which causes muscle wasting, areproduced. The numbers of viral particles increase and the patientdevelops the Acquired Immune Deficiency Syndrome, AIDS, which may last 2to 4 years before the individual's demise. AIDS, therefore, is notsimply a virus infection, although the viral infection is believed to bean integral part of the etiology of the disease.

HIV has a powerful ability to mutate. It is this capability that makesit difficult to create a vaccine or to develop long-term anti-viralpharmaceutical treatments. As more people continue to fail the presentcomplex pharmaceutical regimens, the number of resistant viral strainsis increasing. This is a particularly dangerous pool of HIV and poses aconsiderable threat. These resistant mutants also add to thedifficulties in developing vaccines. This epidemic infection is out ofcontrol, and the widely popularized polypharmaceutical regimens that areaimed only at lowering the number of viruses are proving to be toocomplex, too toxic, too costly, and too narrow. As a result, since theintroduction of protease inhibitors, in combination with AZT-type drugs,increasing numbers of people are failing such therapies. Further, thecontinuing production of free radicals and cytokines, which may becomelargely independent of the virus, perpetuates the dysfunctions of theimmune system, the gastrointestinal tract, the nervous system, and manyother organs in AIDS. The published scientific literature indicates thatmany of these diverse organ system dysfunctions are due to systemic GSHdeficiencies that are engendered by the virus and its free radicals. GSHis consumed in HIV infections because it is the principal, bulwarkantioxidant versus free radicals. An additional cause of erosion of GSHlevels is the presence of numerous disulfide bonds (—S—S—) in HIVproteins, such as the GP-120 discussed above. Disulfide bonds react withGSH and oxidize it.

The current HIV/AIDS pharmaceuticals take good advantage of the conceptof pharmaceutical synergism, wherein two different targets in oneprocess are hit simultaneously. The effect is more than additive. Thedrugs now in use were selected to inhibit two very different points inthe long path of viral replication. The pathway of viral replication canbe depicted simply:

HIV Replication Pathway - - - → - - - → - - - → - - - → - - - → point #1point #2 point #3 point #4 point #5 Virus attacks Virus makes Viral DNAProviral DNA Viral RNA is and enters DNA from is integrated is inactivefor produced, the cell its RNA into cells' a long time, but along withDNA activators will viral mem- start HIV branes, and replicatingproteins, rapidly which are assembled Viral gp120 Reverse Integrase isNF kappa B is Viral protein and transcriptase the enzyme the activatorof protease CD4+ cell is the involved dormant HIV is involved receptorsand enzyme DNA and others are involved glutathione involved levels mustbe low for activation to occur AZT, ddl, Glutathione Protease ddCInhibitors

Point #2 was the earliest point of attack, using AZT-types of drugs,including ddI, ddC and others. These are toxic and eventually virusesbecome resistant to these Reverse Transcriptase inhibitors.

Point #5 is a late replication step, and this is where proteaseinhibitors function. The drug blocks viral protease, an enzyme thatsnips long protein chains to just the right length so the viral coatfits exactly around the nucleic acid core, and that proteins havingdifferent biological activities are separated. By themselves, proteaseinhibitors foster the rapid development of resistant, mutant strains.

By combining Reverse Transcriptase inhibitors plus protease inhibitors,synergism was obtained and the amounts of viral particles in the plasmaplummeted, while the speed of the developing mutant resistant viralstrains was slowed, compared to using only one type of inhibitor. Theinitial promise of these combination therapies or “cocktails” has beentainted by increasing numbers of failures, which are expected to rise asresistant mutants develop, albeit more slowly than the use of the drugsseparately.

New therapies include additional drugs in the classes of ReverseTranscriptase inhibitors and protease inhibitors. Also, drugs are indevelopment to block point #3, wherein the enzyme, integrase, integratesthe HIV DNA into the infected cell's DNA, analogous to splicing it smalllength of wire into a longer wire. Vaccine development also continues,although prospects seem poor because HIV appears to be a moving targetand seems to change as rapidly as a chameleon. Vaccine development isalso impaired by the immune cell affinity of the virus.

Human Immunodeficiency virus-infected individuals have lowered levels ofserum acid-soluble thiols and GSH in plasma, peripheral blood monocytes,and lung epithelial lining fluid. In addition, it has been shown thatCD4+ and CD8+ T cells with high intracellular GSH levels are selectivelylost as HIV infection progresses. This deficiency may potentiate HIVreplication and accelerate disease progression, especially inindividuals with increased concentrations of inflammatory cytokinesbecause such cytokines stimulate HIV replication more efficiently inGSH-depleted cells. GSH and glutathione precursors such as N-acetylcysteine (NAC) can inhibit cytokine-stimulated HIV expression andreplication in acutely infected cells, chronically infected cells, andin normal peripheral blood mononuclear cells.

It is noted that depletion of GSH is also associated with a processesknown as apoptosis, or programmed cell death. Thus, intercellularprocesses that artificially deplete GSH may lead to cell death, even ifthe underlying process itself is not lethal. See:

Arpadi, S. M., Zang, E, Muscat J. and Richie, J.: Glutathione deficiencyin HIV-1-infected children with growth failure, (submitted forpublication).

Baker, D. H. and Wood, R. J.: Cellular antioxidant status and humanimmunodeficiency virus replication. Nutr. Rev. 50: 15-8, 1992.

Baruchel, S., and Wainberg, M. A.: The role of oxidative stress indisease progression in individuals infected by the humanimmunodeficiency virus. J. Leukocyte Biol. 52: 111-114, 1992.

Buhl, R., Holroyd, K. J., Mastrangli, A., Cantin, A. M., Jaffe, H. A.,Wells, F. B., Saltini, C. and Crystal, R. G.: Systemic glutathionedeficiency in symptom-free HIV-seropositive individuals. Lancet ii:1294-1298, 1989.

de Quay, B., Malinverni, R. and Lauterburg, B. H.: Glutathione depletionin HIV-infected patients: role of cysteine deficiency and effect of oralN-acetylcysteine. AIDS 6: 815-9, 1992.

Droge, W., Eck, H. P. and Mihm, S.: HIV-induced cysteine deficiency andT-cell dysfunction—a rationale for treatment with N-acetylcysteine.Immunol. Today 13: 211-4, 1992.

Eck, H. P., Gmunder, H., Hartmann, M., Petzoldt, D., Daniel, V. andDroge, W.: Low concentrations of acid-soluble thiol (cysteine) in theblood plasma of HIV-infected patients. Biol. Chem. Hoppe-Seyler 370:101-108, 1989.

Fauci, A. S.: Multifactorial nature of human immunodeficiency virusdisease: Implications for therapy. Science 262: 1011-1018, 1993.

Foley, P. Kazazi, F., Biti, R., Sorrell, T. C., and Cunningham, A. L.:HIV infection of monocytes inhibits the T-lymphocyte proliferativeresponse to recall antigens via production of eicosanoids. Immunology75: 391-97, 1992.

Hasan, V., Thomas, D., Aclami, J. et al. : Stimulation of a human T-cellclone with anti-CD3 or tumor necrosis factor induces NFkB translocationbut not human immunodeficiency virus 1 enhancer-dependent transcription.Proc. Natl. ACAD. sCI. 87: 7861-65, 1990.

Ho, W. Z. and Douglas, S. D.: Glutathione and N-acetylcysteinesuppression of human immunodeficiency virus replication in humanmonocyte/macrophages in vitro. AIDS Res. Hum. Retroviruses, 8: 1249-53,1992.

Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L.:Redox status of cells influences constitutive or induced NF?Btranslocation and HIV long terminal repeat activity in human T andmonocytic cell lines. J. Immunol. 149: 3386-93, 1992.

Kobayashi, S., Hamamoto, Y., Kobayashi, N., and Yamamoto, N.: Serumlevel of TNFa in HIV-infected individuals. AIDS 4: 169 1990.

Kalebic, T., Kinter, A., Poli, G., Anderson, M. E., Meister, A. andFauci, A. S.: Suppression of human immunodeficiency virus expression inchronically infected monocytic cells by glutathione, glutathione ester,and N-acetylcysteine. Proc. Natl. Acad. Sci. USA 87: 986-990, 1991.

LeGrand-Poels, S., Vaira, D., Pincemail, J., Van de Vorst, A. andPiette, J.: Activation of human immunodeficiency virus type 1 byoxidative stress. AIDS Res. Hum. Retrov. 6: 1389-97, 1990.

Mihm, S., Ennen, J., Pessara, U., Kurth, R. and Droge, W.: Inhibition ofHIV-1 replication and NF-kb activity by cysteine and cysteinederivatives. AIDS 5: 497-503, 1991.

National Institutes of Health. Dr. Howard C. Greenspan. Chairman ofConference on Free Radicals and Antioxidants in HIV/AIDS, Nov. 12-13,1993/Greenspan, H. C. The role of reactive oxygen species, antioxidantsand phytopharmaceuticals in human immunodeficiency virus activity.Med-Hypotheses 40: 85-92, 1993.

Roederer, M., Raju, P. A., Staal, F. J. T., Herzenberg, L. A. andHerzenberg, L. A.: N-acetylcysteine inhibits latent HIV expression inchronically infected cells. AIDS Res. Human Retrovir. 7: (6) 563-567,1991.

Roederer, M., Staal, F. J. T., Osada, H., Herzenberg, L. A. andHerzenberg, L. A.: CD4 and CD8 T cells with high intracellularglutathione levels are selectively lost as the HIV infection progresses.Internat. Immunol. 3: 933-37, 1991.

Roederer, M., Staal, F. J. T., Raju, P. A., Ela, S. W., Herzenberg, L.A. and Herzenberg, L. A.: Cytokine-stimulated human immunodeficiencyvirus replication is inhibited by N-acetyl-L-cysteine. Proc. Natl. Acad.Sci. USA 87: 4884-4888, 1990.

Schreck, R. Rieber, P., and Baeurle, P. A.: Reactive oxygenintermediates as apparently widely used messengers in the activation ofthe NF-kb transcription factor and HIV-1. EMBO J. 10: 2247-2258, 1991.

Staal, F. J., Roederer, M., Herzenberg, L. A. and Herzenberg, L. A.:Glutathione and immunophenotypes of T and B lymphocytes in HIV-infectedindividuals. Ann. NY Acad. Sci. 651: 453-63, 1992.

Staal, F. J. T., Roederer, M. Herzenberg, L. A., and Herzenberg, L. A.:Intracellular thiols regulate activation of nuclear factor kappa-B andtranscription of human immunodeficiency virus. Proc. Natl. Acad. Sci.USA 87: 9943-9947, 1990.

Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., Herzenberg, L.A. and Herzenberg, L. A.: Glutathione deficiency and humanimmunodeficiency virus infection. Lancet 339: 909-12, 1992.

Staal, F. J., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A.,McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A.,Herzenberg, L. A. and Herzenberg, L. A.: Intracellular glutathionelevels in T cell subsets decrease in HIV-infected individuals. AIDS Res.Hum. Retroviruses 8: 305-11, 1992.

Staal, F. J. T., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W.,Herzenberg, L. A., and Herzenberg, L. A.: Antioxidants inhibitstimulation of HIV transcription. AIDS Res. Hum. Retrov. 9: 299-306,1993.

Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Harel-Bellan,A. and Farrar, W. L.: Human immunodeficiency virus glycoprotein (gp120)induction of monocyte arachidonic acid metabolites and interleukin 1.Proc. Natl. Acad. Sci. 86: 621-625, 1989.

2) Diabetes Mellitus

Diabetes mellitus is found in two forms, childhood or autoimmune (typeI, IDDM) and late-onset or non-insulin dependent (type II, NIDDM). Theformer constitute about 30% and the remainder represent the bulk ofcases seen. Onset is generally sudden for Type I, and insidious for TypeII. Symptoms include excessive urination, hunger and thirst with a slowsteady loss of weight in the first form. Obesity is often associatedwith the second form and has been thought to be a causal factor insusceptible individuals. Blood sugar is often high and there is frequentspilling of sugar in the urine. If the condition goes untreated, thevictim may develop ketoacidosis with a foul-smelling breath similar tosomeone who has been drinking alcohol. The immediate medicalcomplications of untreated diabetes can include nervous system symptoms,and even diabetic coma.

Because of the continuous and pernicious occurrence of hyperglucosemia(very high blood sugar levels), a non-enzymatic chemical reaction occurscalled glycation. Since glycation occurs far more frequently insidecells, the inactivation of essential enzyme proteins happens almostcontinually. One of the most critical enzymes, γ-glutamyl-cysteinesynthetase, is glycated and readily inactivated. This enzyme is thecrucial step in the biosynthesis of glutathione in the liver.

The net result of this particular glycation is a deficiency in theproduction of GSH in diabetics. Normally, adults produce 8-10 gramsevery 24 hours, and it is rapidly oxidized by the cells. GSH is in highdemand throughout the body for multiple, essential functions, forexample, within all mitochondria, to produce chemical energy called ATP.Brain cells, heart cells, and others simply will not function well andcan be destroyed through apoptosis.

GSH is the major antioxidant in the human body and the only one we areable to synthesize, de novo. It is also the most common small molecularweight thiol in both plants and animals. Without GSH, the immune systemcannot function, and the central and peripheral nervous systems becomeaberrant and then cease to function. Because of the dependence on GSH asthe carrier of nitric oxide, a vasodilator responsible for control ofvascular tone, the cardiovascular system does not function well andeventually fails. Since all epithelial cells seem to require GSH, theintestinal lining cells don't function properly and valuablemicronutrients are lost, nutrition is compromised, and microbes aregiven portals of entry to cause infections.

The use of GSH precursors cannot help to control the GSH deficiency dueto the destruction of the rate-limiting enzyme by glycation. As GSHdeficiency becomes more profound, the well-known sequellae of diabetesprogress in severity. The complications described below are essentiallydue to runaway free radical damage since the available GSH supplies indiabetics are insufficient.

Ceriello, A., Giugliano, D., Quatraro, A. & Lefebvre, P. J.:Anti-oxidants show an anti-hypertensive effect in diabetic andhypertensive subjects. Clin. Sci. 81: 739-742, 1991.

Paolisso, G., Giugliano, D., Pizza, G., Gambardella, A., Tesauro, P.,Varricchio, M. & D'Onofrio, F.: Glutathione infusion potentiatesglucose-induced insulin secretion in aged patients with impaired glucosetolerance. Diabetes Care 15: 1-7, 1992.

Reducing sugars are known to interact with free amino groups inproteins, lipids, and nucleic acids to form Amadori product and producereactive oxygen species through the glycation reaction. Under diabeticconditions, glucose level is elevated and the glycated proteinsincreased. Cu,Zn-SOD has been shown to be glycated and inactivated underdiabetic conditions and that ROS produced from the Amadori productcaused site-specific fragmentation of Cu,Zn-SOD. Fructose, which isproduced through polyol pathway, has stronger glycating capacity thanglucose because the physiologic proportion of the linear form is higherthan that of cyclized form. Fructose, as well as ribose, can bring aboutapoptosis in pancreatic β islet cell line. Levels of intracellularperoxides, protein carbonyls, and malondialdehyde are increased in thepresence of fructose. In addition, methylglyoxal and 3-deoxyglucosonehave also been shown to induce apoptotic cell death. 3-Deoxyglucosone, a2-oxoaldehyde, is produced through the degradation of Amadori compounds.Both compounds are elevated during hyperglycemia and accelerate theglycation reaction. These compounds are toxic to cells, due to theirhigh reactivity, and a scavenging system with NADPH-dependent reducingactivity exists, including aldehyde reductase. Junichi Fujii and NaoyukiTaniguchi, Dysfunction of Redox System by Reactive Oxygen Species,Nitric Oxide and the Glycation Reaction: A Possible Mechanism forApoptotic Cell Death (Poster), Proceedings of 3rd Internet WorldCongress on Biomedical Sciences, 1996, 12, 9-20 Riken, Tsukuba, Japan.See, also:

Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. 1996. Involvement ofMACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNFreceptor-induced cell death. Cell 85: 803-815.

Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, et al. 1994.Suppression of antioxidative enzyme expression by transforming growthfactor-b1 in rat hapatocytes J. Biol. Chem. 269: 15488-15492.

Rosen D R, Siddique T, Patterson D, Figlewicz D A, Sapp P. et al. 1993.Mutations in Cu/Zn-superoxide dismutase gene are associated withfamilial amyotrophic lateral sclerosis. Nature 362: 59-62.

Fujii J, Myint T, Seo H G, Kayanoki Y, Ikeda Y, et al. 1995.Characterization of wild-type and amyotrophic lateral sclerosis-relatedmutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infectedinsect cells. J. Neurochem. 64: 1456-1461.

Deng H-X, Hentati A, Tainer J A, Iqbal Z, Cayabyab A, et al. 1993.Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxidedismutase. Science 261: 1047-1051.

Rothstein J D, Bristol L A, Hosler B, Brown R H, Jr, Kuncl R W, 1994.Chronic inhibition of superoxide dismutase produces apoptotic death ofspinal neurons. Proc. Natl. Acad. Sci. U.S.A. 91: 4155-4159.

Gurnery M E, Pu H, Chiu A Y, Dal Canto, M C, Polchow C Y, et al. 1994.Motor neuron degradation in mice that express a human Cu,Zn-superoxidedismutase mutation. Science 264: 1772-1775.

Hockenbery D M, Oltvai Z N, Yin X-M, Milliman C L, Korsmeyer S J, 1993.Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241-251.

Kayanoki Y, Fujii J, Islam K N, Suzuki K, Kawata S, et al. 1996. Theprotective role of glutathione peroxidase in apoptosis induced byreactive oxygen species. J. Biochem. 119: 817-822.

Islam K N, Kayanoki Y, Kaneto H, Suzuki K, Asahi M, et al. 1996. TGF-b1triggers oxidative modifications and enhances apoptosis in HIT cellsthrough accumulation of reactive oxygen species by suppression ofcatalase and glutathione peroxidase. Free Radic. Biol. Med. in press.

Taniguchi N. 1992. Clinical significances of superoxide dismutases:Changes in aging, diabetes, ischemia, and cancer. Adv. Clin. Chem. 29:1-59.

Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, et al. 1987.Glycation and inactivation of human Cu—Zn-superoxide dismutase.Identification of the in vitro glycation sites. J. Biol. Chem. 262:16969-16972.

Ookawara T, Kawamura N, Kitagawa Y, Taniguchi N. 1992. Site-specific andrandom fragmentation of Cu,Zn-superoxide dismutase by glycationreaction. Implication of reactive oxygen species. J. Biol. Chem. 267:18505-18510.

Fujii J, Mint T, Okado A, Kaneto H, Taniguchi N. 1996. Oxidative stresscaused by glycation of Cu,Zn-superoxide dismutase and its effects onintracellular components. Nephrol. Dial. Transplant (Supple 19) inpress.

Kaneto H, Fujii J, Myint T, Islam K N, Miyazawa N, et al. 1996. Reducingsugar triggers oxidative modification and apoptosis in pancreaticb-cells by provoking oxidative stress through glycation reaction.Biochem. J. in press.

Okado A, Kawasaki Y, Hasuike Y, Takahashi M, Teshima T, et al. 1996.Induction of apoptotic cell death by methylglyoxal and 3-deoxyglucosonein macrophage-derived cell lines. Biochem. Biophys. Res. Commun. 225:219-224.

Takahashi M, Fujii J, Teshima T, Suzuki K, Shiba T, et al. 1995.Identity of a major 3-deoxyglucosone-reducing enzyme with aldehydereductase in rat liver established by amino acid sequencing and cDNAexpression. Gene 127: 249-253.

Takahashi M, Lu Y, Myint T, Fujii J, Wada Y, et al. 1995. In vivoglycation of aldehyde reductase, a major 3-deoxyglucosone reducingenzyme. Identification of glycation sites. Biochemistry 34: 1433-1438.

Takahashi M, Fujii J, Miyoshi E, Hoshi A, Taniguchi N. 1996. Elevationof aldose reductase gene expression in rat primary hepatoma and hepatomacell lines: Implication in detoxification of cytotoxic aldehydes. Int.J. Cancer. 87: 337-341.

Seo H G, Takata I, Nakamura M, Tatsumi H, Suzuki K, et al. 1995.Induction of nitric oxide synthase and concomitant suppression ofsuperoxide dismutases in experimental colitis in rats. Arch. Biochem.Biophys. 324: 41-47.

Kaneto H, Fujii J, Seo H G, Suzuki K, Matsuoka M, et al. 1995. Apoptoticcell death triggered by nitric oxide in pancreatic b-cells. Diabetes 44:733-738.

Asahi M, Fujii J, Suzuki K, Seo H G, Kuzuya T, et al. 1995. Inactivationof glutathione peroxidase by nitric oxide. Implication for ctyotoxicity.J. Biol. Chem. 270: 21035-21039

Cell-cell adhesion is critical in generation of effective immuneresponses and is dependent upon the expression of a variety of cellsurface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) andvascular cell adhesion molecule (VCAM-1: CD 106) are inducible cellsurface glycoproteins. The expression of these surface proteins areknown to be induced in response to activators such as cytokines (TNF-α,IL-1 α & β), PMA, lipopolysaccharide and oxidants. The ligands forICAM-1 and VCAM-1 on lymphocyte are LFA-1 (CD11a/CD18) and VLA-4,respectively. The inappropriate or abnormal sequestration of leukocytesat specific sites is a central component in the development of a varietyof autoimmune diseases and pathologic inflammatory disorders. Focalexpression of ICAM-1 have been reported in arterial endotheliumoverlying early foam cell lesions in both dietary and genetic models ofatherosclerosis in rabbits. A role of VCAM-1 in the progression ofcoronary lesions has also been suggested. Loss or gain of cell surfacemolecules is thought to determine the mobilization, emigration andinvasiveness of epithelial cancer cells. Monocytes from patients withdiabetes mellitus are known to have increased adhesion to endothelialcells in culture. Regulation of adhesion molecule expression andfunction by reactive oxygen species via specific redox sensitivemechanisms have been reported. Antioxidants can block induced adhesionmolecule expression and cell-cell adhesion. Sashwati Roy and Chandan K.Sen. Adhesion Molecules And Cell-Cell Adhesion,http://packer.berkeley.edu/research/Cell/adhes.

The diabetic will become more susceptible to infections because theimmune system approaches collapse when GSH levels fall, analogous tocertain defects seen in HIV/AIDS. Peripheral vasculature becomescompromised and blood supply to the extremities is severely diminishedbecause GSH is not available in sufficient amounts to stabilize thenitric oxide (.NO) to effectively exert its vascular dilation(relaxation) property. Gangrene is a common sequel and successiveamputations are often the result in later years.

Peripheral neuropathies, the loss of sensation commonly of the feet andlower extremities develop, often followed by aberrant sensations likeburning or itching, which can't be controlled. Retinopathy andnephropathy are later events that are actually due to microangiopathy,excessive budding and growth of new blood vessels and capillaries, whichoften will bleed due to weakness of the new vessel walls. This bleedingcauses damage to the retina and kidneys with resulting blindness andrenal shutdown, the latter results in required dialysis. Cataracts occurwith increasing frequency as the GSH deficiency deepens.

Large and medium sized arteries become sites of accelerated, severeatherosclerosis, with myocardial infarcts at early ages, and of a moresevere degree. If diabetics go into heart failure, their mortality ratesat one year later are far greater than in non-diabetics. Further, ifcoronary angioplasty is used to treat their severe atherosclerosis,diabetics are much more likely to have renarrowing of cardiac vessels,termed restenosis.

The above complications are due, in large measure, to GSH deficiency andongoing free radical reactions. These sequellae frequently andeventually occur despite the use of insulin injections daily that lowerblood sugar levels. Good control of blood sugar levels is difficult forthe majority of diabetics.

3) Macular Degeneration

Approximately 1 million people in the United States have significantmacular degeneration. One out of every 4 persons aged 55 or above nowhas maculir degeneration and 1 in 2 above the age of 80. As ourpopulation ages, this principal cause of blindness in the elderly willincrease as well. By the year 2002, 15 million people in the U.S. willsuffer from macular degeneration.

Age-related macular degeneration (ARMD) is the disease characterized byeither a slow (dry form) or rapid (wet form) onset of destruction andirrevocable loss of rods and cones in the macula of the eye. The maculais the approximate center of the retina wherein the lens of the eyefocuses its most intense light. The visual cells, known as the rods andcones, are an outgrowth and active part of the central nervous system.They are responsible and essential for the fine visual discriminationrequired to see clear details such as faces and facial expression,reading, driving, operation of machinery and electrical equipment andgeneral recognition of surroundings. Ultimately, the destruction of therods and cones leads to functional, legal blindness. Since there is noovert pain associated with the condition, the first warnings of onsetare usually noticeable loss of visual acuity. This may already signallate stage events. It is now thought that one of the very first eventsin this pathologic process is the formation of a material called“drusen”.

Drusen first appears as either patches or diffuse drops of yellowmaterial deposited upon the surface of the retina in the macula lutea oryellow spot. This is the area of the retina there sunlight is focused bythe lens. It is the area of the retina that contains the highest densityof rods for acuity. Although cones, which detect color, are lost as wellin this disease, it is believed to be loss of rods that causes theblindness. Drusen has been chemically analyzed and found to be composedof a mixture of lipids, much of which are peroxidized by free radicalreactions. The Drusen first appears as small collections of material atthe base of Bruch's membrane. This produces “bubbles” which push thefirst layer of cells up off the membrane. Vascular budding, neovasculargrowth, first appears in these channels.

This first layer of cells is unique. They are retinal pigmentedepithelial (RPE) cells and these cells are distantly related to CNSmicroglia and have a phagocytic function. They are also the layer ofcells immediately below the primary retinal cells, the rods and cones.The RPE cells are believed to serve a protective function for the rodsand cones since they consume the debris cast off by the rods and cones.It is not known yet whether the pigmented material serves a protectivefunction or is related to phagocytosis only. However, this pigment,although concentrated in organelles, is believed to be composed ofperoxidized lipids and melanin.

It is believed, because of the order of events in model systems, thatthe loss of RPE cells occurs first in ARMD (Age Related MacularDegeneration). Once an area of the retinal macula is devoid of RPEcells, loss of rods, and eventually some cones, occurs. Finally, buddingof capillaries begins and we see the typical microangiopathy associatedwith late stage ARMD. It is also known that RPE cells require largequantities of GSH for their proper functioning. When GSH levels dropseverely in these cells, in cell cultures where they can be studied,these cells begin to die. When cultures of these cells are supplementedwith GSH in the medium, they thrive. There is increasing evidence thatprogression of the disease is paced by a more profound deficiency in GSHwithin the retina and probably within these cells, as indicated by cellculture studies.

It is generally believed that “near” ultraviolet (UVB) and visual lightof high intensity primarily from sunlight is a strong contributingfactor of ARMD. People with light-colored irises constitute a populationat high risk, as do those with jobs that leave them outdoors and inequatorial areas where sunlight is most intense. Additional free radicalinsults, like smoking, add to the risk of developing ARMD.

Several approaches have been recently tested, including chemotherapy,without success. Currently, there is no effective therapy to treat ARMD.Laser therapy has been developed which has been used widely to slow thedamage produced in the slow onset form of the disease by cauterizingneovascular growth. However the eventual outcome of the disease, once ithas started to progress, is certain.

4) Cellular Regulation by Reactive Oxygen Species

There are a number of types of messengers carrying signals betweencells. One type of messenger which has received significant attentionrecently are small molecule oxidative or free radical agents, whichinclude reactive oxygen species (ROS). These messengers often act by anon-specific interaction with biological macromolecules which may resultin a change in configuration. For example, protein secondary structureis typically controlled by cysteine residues, which are susceptible tooxidation with the formation of disulfide bonds. Oxidization of thesebonds forming linkages may result in substantial changes in proteinconfiguration and function.

It has thus become increasingly apparent that O₂ ⁻ and H₂O₂ aresignaling molecules, changing the behavior of proteins as diverse astranscription factors and membrane receptors by virtue of their abilityto undergo redox reactions with the proteins with which they interact,converting —SH groups to disulfide bonds, for example, and changing theoxidation states of enzyme-associated transition metals. As signalingmolecules, O₂ ⁻ and H₂O₂ are manufactured by several types of cells,including fibroblasts, endothelial and vascular smooth muscle cells,neurons, ova, spermatozoa and cells of the carotid body. All these celltypes appear to use an NAD(P)H oxidase similar to the classicalleukocyte NADPH oxidase to produce these oxidants. The stimuli thatelicit oxidant production, however, and the purposes for which theoxidants are employed, vary from cell to cell.

Fibroblasts manufacture small but significant amounts of O₂ ⁻ inresponse to inflammatory mediators such as N-formylated peptides andinterleukin-1. The O₂ ⁻ produced by these cells has been postulated tofunction as a signaling molecule. Optical spectroscopy has shown thatfibroblast membranes contain a heme protein that is different from theflavocytochrome subunit of the leukocyte NADPH oxidase but hasproperties very similar to those of the leukocyte protein. This hemeprotein has been suggested as the source of the O₂ ⁻ made by thesecells.

Endothelial and vascular smooth muscle cells use an NAD(P)H oxidase toproduce O₂ ⁻ in response to angiotensin II, a peptide hormone thatincreases blood pressure. This increase in blood pressure appears to bedue to the consumption by O₂ ⁻ of the NO. that is generated on acontinuing basis by the endothelial cells. The resulting fall in NO.concentration raises blood pressure by attenuating or eliminating thevasodilatory effect of NO. that normally prevails in the vascular tree.

Neuronal cells in culture produce oxidants when exposed to amyloidβ-peptide, found in amyloid deposits seen in the brains of patients withAlzheimer's disease, or related peptides from other amyloid diseases.The possibility that this O₂ ⁻ is produced by an NADPH oxidase issuggested by the observation that flavoprotein inhibitors known to acton the leukocyte NADPH oxidase also inhibit oxidant production in thissystem. The production of oxidants may be part of a defense used by theneuron against the peptide, with these oxidants perhaps reacting withthe peptide to render it susceptible to proteolytic cleavage.

At the moment of fertilization, a membrane NADPH oxidase in sea urchinova is activated to produce large amounts of H₂O₂. This oxidantcross-links the proteins of the fertilization membrane by formingdityrosyl bridges, making the membrane impermeable to spermatozoa andthereby preventing polyspermy. This mechanism is common to other species

O₂ ⁻ appears to be necessary for the normal function of spermatozoa.When stimulated by a calcium ionophore, normal spermatozoa generate a 3-to 5-min burst of O₂. The O₂ ⁻ produced in this reaction is involved incapacitation of the spermatozoa, because the acrosomal response to anumber of stimuli is suppressed by superoxide dismutase. On the otherhand, spermatozoa that produce O₂ ⁻ without stimulation are functionallyabnormal, perhaps because of a generalized disruption in their signalingmachinery.

The carotid body is a small organ located at the bifurcation of thecommon carotid artery that measures the oxygen tension of the blood.This organ manufactures H₂O₂ on a continuing basis, and immunologicalanalysis has shown that its cells contain all 4 of the specific subunitsof the leukocyte NADPH oxidase, or proteins very closely related tothose subunits. It has been postulated that a carotid body NADPH oxidasevery similar or identical to the leukocyte NADPH oxidase is a keycomponent of the oxygen-measuring apparatus of the carotid body.

Thus, in addition to phosphorylation as a control mechanism overregulatory protein configuration and function, reactive oxygen speciesmay also play an important role in cellular regulation and signaling.Selective cysteine oxidation-reduction also serves as an importantmechanism for post-translational modification of protein function. Thismechanism, termed “redox regulation”, has been implicated in a varietyof cellular processes such as DNA synthesis, enzyme activation, geneexpression, and cell cycle regulation.

Thioredoxin (TRX) is a pleiotropic cellular factor which hasthiol-mediated redox activity and plays important roles in regulation ofcellular processes, including gene expression. TRX exists either in areduced, or oxidized form and participates in redox reactions throughthe reversible oxidation of this active center dithiol. Activity of anumber of transcription factors is post-translationally altered by redoxmodification(s) of specific cysteine residue(s). One such factor isNF-κB, whose DNA-binding activity is altered by TRX treatment in vitro.The DNA-binding activity of AP-1 is modified by a DNA repair enzyme,Redox Factor-1 (Ref-1). Ref-1 activity is in turn modified by variousredox-active compounds, including TRX. TRX translocates from thecytoplasm into the nucleus in response to PMA treatment to associatedirectly with Ref-1 and modulates not only the DNA-binding but also thetranscriptional activity of the AP-1 molecule.

Human thioredoxin (hTRX) has thus been shown to be an important redoxregulator in those biological processes. hTRX can function directly byinteracting with the target molecules such as NF-κB transcriptionfactor, or indirectly via another redox protein known as redox factor 1(Ref-1). Structural Basis Of Thioredoxin-Mediated Redox-Regulation, Qinet al, (poster), Proceedings of 3rd Internet World Congress onBiomedical Sciences, 1996, 12, 9-20 Riken, Tsukuba, Japan. See, also:

Abate, C., Patel, L., Rauscher III, R. J., and Curran, T. (1990) Redoxregulation of Fos and Jun DNA binding activity in vitro. Science 249,1157-1161.

Baeuerle, P. A., and Henkel, T. Function and activation of NF-kB in theimmune system. (1994) Annu. Rev. Immunol. 12, 141-179.

Bax and Grzesiek, S. (1993) Methodological advances in protein NMR.Accounts Chem. Res. 26, 131-138.

Beg, A. A., and A. S. Baldwin, Jr. The IkB proteins: multifunctionalregulatorsof Rel/NF-kB transcription factors. (1993) Genes and Dev, 7,2064-2070.

Clore, G. M. and Gronenborn, A. M. (1991) Structures of larger proteinsin solution: three- and four-dimensional heteronuclear NMR spectroscopy.Science 252, 1390-1399.

Gilmore, T. D., and Morin, P. J. The IkB proteins: members of a multifunctional family. (1993) Trends Genet. 9, 427-433.

Ghosh, S., van Duyne, G., Ghosh, S., and Sigler, P. Structure of NF-kBp50 homodier bound to a kB site. (1995) Nature 373, 303-310.

Hayashi, T., Ueno, Y., and Okamoto, T. Oxidoredictive regulation ofnuclear factor kB. (1993) J. Biol. Chem. 268 (15): 11380-11388.

Holmgren, A. (1989) Thioredoxin and glutaredoxin. J. Biol. Chem. 264,13963-13966.

Liou, H.-C., and Baltimore, D. Regulation of the NF-kB/rel transcriptionfactor and IkB inhibitor system. (1993) Curr. Opin. Cell. Biol. 5,477-487.

Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R.T. Thioredoxin regulates the DNA binding activity of NF-kB by reductionof a disulfide bond involving cysteine 62. (1992) Nucleic AcidsResearch, 20 (15): 3821-3830.

Muller, C. W., Rey, F. A., Sodeoka, M., Verdine, G. L., and Harrison, S.C. Structure of the NF-kB p50 homodimer bound to DNA. (1995) Nature 373,311-317.

Powis, G., Briehl, M., and Oblong, J. (1995) Redox signaling and thecontrol of cell growth and death. Pharmac. Ther. 68, No. 1, 149-173.

Qin, J., Clore, G. M., Kennedy, W M P, Huth, J., and Gronenborn, A. M.(1995) Solution structure of human thioredoxin in a mixed disulfideintermediate complex with its target peptide from the transcriptionfactor NFkB. Structure, 15: 3, 289-297.

Qin, J., Clore, G. M., Kennedy, W M P, and Gronenborn, A. M. Thesolution structure of human thioredoxin complexed with its target fromRef-1 reveals peptide chain reversal. (1996b) Structure, 4 (5), 613-620.

Walker, L., Robson, C. N., Black, E., Gillespie, D., and Hickson, I.(1993) Identification of residues in the human DNA repair enzyme HAP1(Ref-1) that are essential for redox regulation of Jun DNA binding. Mol.Cell. Biol. 13, 5370-5376.

Xanthoudakis, S., Miao, G. G., Wang, F., E. Pan, Y., and Curran, T.(1992) Redox activation of Fos-Jun DNA binding activity is mediated by aDNA repair enzyme. EMBO J. 11, 653-656.

Xanthoudakis, S., Miao, G. G., and Curran, T. (1994) The redox andDNA-repair activities of Ref-1 are encoded by nonoverlapping domains.Proc. Natl. Acad. Sci. USA, 91, 23-27.

Cellular redox status modulates various aspects of cellular eventsincluding proliferation and apoptosis. TRX is a small (13 kDa),ubiquitous protein with two redox-active half-cystine residues in anactive center, -Trp-Cys-Gly-Pro-Cys-, and is also known as adult T-cellleukemia-derived factor (ADF) involved in HTLV-I leukemogenesis. Thepathway for the reduction of a protein disulfide by TRX entailsnucleophilic attack by one of the active-site sulfhydryls to form aprotein-protein disulfide followed by intramolecular displacement of thereduced target proteins with concomitant formation of oxidized TRX.Besides the activity as an autocrine growth factor for HTLV-I-infected Tcells and Epstein-Barr virus-transformed lymphocytes, numerous studieshave shown the importance of ADF/TRX as a cellular reducing catalyst inhuman physiology.

In vitro and in vivo experiments showed that TRX augmented theDNA-binding and transcriptional activities of the p50 subunit of NF-κBby reducing Cys 62 of p50. Direct physical association of TRX and anoligopeptide from NF-κB p50 has been revealed by NMR study in vitro.Redox regulation of Jun and Fos molecules has also been implicated.Various antioxidants strongly activate the DNA-binding andtransactivation abilities of AP-1 complex. TRX enhances the DNA-bindingactivity of Jun and Fos, in a process which requires other molecules,such as redox factor-1 (Ref-1).

NF-κB regulates expression of a wide variety of cellular and viralgenes. These genes include cytokines such as IL-2, IL-6, IL-8, GM-CSFand TNF, cell adhesion molecules such as ICAM-1 and E-selectin,inducible nitric oxidase synthase (iNOS) and viruses such as humanimmunodeficiency virus (HIV) and cytomegalovirus. Through the causalrelationship with these genes, NF-κB is considered to be causallyinvolved in the currently intractable diseases such as acquiredimmunodeficiency syndrome (AIDS), hematogenic cancer cell metastasis andrheumatoid arthritis (RA). Although the genes induced by NF-κB arevariable according to the context of cell lineage and are also under thecontrol of the other transcription factors. NF-κB plays a major role inregulation of these genes and thus contributes a great deal to thepathogenesis. Therefore, biochemical intervention of NF-κB shouldconceivably interfere the pathogenic process and would be effective forthe treatment.

NF-κB consists of two subunit molecules, p65 and p50, and usually existsas a molecular complex with an inhibitory molecule, IκB, in the cytosol.Upon stimulation of the cells such as by proinflammatory cytokines, IL-1and TNF, IκB is dissociated and NF-κB is translocated to the nucleus andactivates expression of target genes. Thus activity of NF-κB itself isregulated by the upstream regulatory mechanism. Not much is know aboutthe upstream signaling cascade. However, there are at least twoindependent steps in the NF-κB activation cascade: kinase pathways andredox-signaling pathway. These two distinct pathways are involved in theNF-κB activation cascade in a coordinate fashion, which may contributeto a fine tune, as well as fail-safe, regulation of NF-κB activity.

At least two distinct types of kinase pathways are known to be involvedin NF-κB activation: NF-κB kinase and IκB kinase. NF-κB kinase is a 43kD serine kinase, associated with NF-κB. This kinase phosphorylates bothsubunits of NF-κB and dissociates it from IκB. There is another kinaseor kinases that is known to phosphorylate IκB. Consistent with thesefindings, NF-κB was shown to be phosphorylated in some cell lines andIκB was phosphorylated in others in response to stimulation with TNF orIL-1. In most of the cases, NF-κB dissociation by kinase cascade is aprimary step of NF-κB activation.

After dissociation from IκB, however, NF-κB must go through the redoxregulation by cellular reducing catalyst, thioredoxin (TRX). TRX isknown to participate in redox reactions through reversible oxidation ofits active center dithiol to a disulfide. Human TRX has been initiallyidentified as a factor responsible for induction of the a subunit ofinterleukin-2 receptor which is now known to be under the control ofNF-κB. It is known that NF-κB can not bind to the κB DNA sequence of thetarget genes until it is reduced.

NF-κB appears to have a novel DNA-binding structure called beta-barrel,a group of beta sheets stretching toward the target DNA. There is a loopin the tip of the beta barrel structure that intercalates with thenucleotide bases and is considered to make a direct contact with theDNA. This DNA-binding loop contains the cystein 62 residue of NF-κB thatis likely the target of redox regulation as a proton donor from TRX. Aboot-shaped hollow on the surface of TRX containing the redox-activecysteines could stably recognize the DNA-binding loop of p50 and islikely to reduce the oxidized cysteine by donating protons in astructure-dependent way. Therefore, the reduction of NF-κB by TRX isconsidered to be specific.

Not much is known about the initiation of the NF-κB signaling cascades.However, pretreatment of cells with antioxidants such asN-acetyl-cysteine (NAC) or a-lipoic acid blocks NF-κB. NAC can alsoblock the induction of TRX. Therefore, anti-NF-κB actions ofantioxidants are considered to be two-fold: 1) blocking the signalingimmediately downstream of the signal elicitation, and 2) suppression ofinduction of the redox effector TRX. It is noted that, in mammalswithout chroic deseases, such as HIV infection, diabetes, etc. whichmight impair physiologic glutathione metabolism, a strategy for thepharmaceutical administration of other antioxidants which improveglutathione metabolism or compounds which are themselves appropriateantioxidants may be employed. It is noted that NAC has been shown tohave certain neurological toxicity in chronic administration, andtherefore this compound is likely inappropriate. On the other hand,lipoic acid may be an advantageous antioxidant alone, or in combinationwith glutathione. Because of the sensitivity of glutathione oraladministration to the particular method of administration, alpha-lipoicacid may have to be administered separately.

The intracellular redox cascade involves successive reduction of oxygenby addition of four electrons and redox regulation of a target protein.Among these ROI hydrogen peroxide has a longest half-life and isconsidered to be a mediator of oxidative signal. On the other hand,cellular reducing system such as TRX counteracts the action of hydrogenperoxide. The intensity of the oxidative signal may be modulated by theinternal GSH level. Similarly, total GSH/GSSG content may influence theresponsiveness of the cellular redox signaling. Therefore, intracellularcycteine required to produce GSH.

See:

Holmgren, A. Ann. Rev. Biochem. 54, 237-271 (1985).

Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J. & Hay, R. T.Nucleic Acids Res. 20, 3821-30 (1992).

Okamoto, T., et al. Int. Immunol. 4, 811-9 (1992).

Abate, C., Patel, L., Rauscher, F. J. III. & Curran, T. Science 249,1157-61 (1990).

Xanthoudakis, S. & Curran, T. Methods Enzymol. 234, 163-74 (1994).

Xanthoudakis, S. & Curran, T. EMBO J. 11, 653-65 (1992).

Pahl, H. L. & Baeuerle, P. A. BioEaays 16, 497-502 (1994).

Holmgren, A. J. biol. Chem. 264, 13963-1366 (1989).

Tagaya, Y., et al. EMBO J. 8, 757-764 (1989).

Yodoi, J. & Uchiyama, T. Immunol. Today 13, 405-11 (1992).

Silberstein, D. S., McDonough, S., Minkoff, M. S. & Balcewicz Sablinska,M. K. J. biol. Chem. 268, 9138-42 (1993).

Iwata, S., et al. J. Immunol. 152, 5633-42 (1994).

Biguet, C., et al. J. biol. Chem. 269, 28865-70 (1994).

Qin, J., Clore, G. M., Kennedy, W. M. P., Huth, J. R. & Gronenborn, A.M. Structure 3, 289-297 (1995).

Meyer, M., Schreck, R. & Baeuerle, P. A. EMBO J. 12, 2005-2015 (1993).

Xanthoudakis, S., Miao, G. G. & Curran, T. Proc. natl. Acad. Sci. U.S.A.91, 23-7 (1994).

Isoda, K. & Nüsslein-Volhard, C. Proc. natl. Acad. Sci. U.S.A. 91,5350-5354 (1994).

Kishigami, S., Kannaya, E., Kikuchi, M. & Ito, K. J. biol. Chem. 270,17072-17074 (1995).

Oblong, J. E., Berggren, M., Gasdaska, P. Y. & Powis, G. J. biol. Chem.269, 11714-20 (1994).

Tonissen, K., et al. J. biol. Chem. 268, 22485-9 (1993).

Forman Kay, J. D., Clore, G. M. & Gronenborn, A. M. Biochemistry 31,3442-52 (1992).

Sadowski, I. & Ptashne, M. Nucleic Acids Res. 17, 7539 (1989).

Perlmann, T., Rangarajan, P. N., Umesono, K. & Evans, R. M. Genes &Develop. 7, 1411-1422 (1993).

Angel, P., et al. Mol. Cell. Biol. 7, 2256-2266 (1987).

Barzilay, G. & Hichson, I. D. BioEssays 17, 713-719 (1995).

Okuno, H., et al. Oncogene 8, 695-701 (1993).

Chida, K. & Vogt, P. K. Proc. natl. Acad. Sci. U.S.A. 89, 4290-4294(1992).

Ng, L., Forrest, D. & Curran, T. Nucleic Acids Res. 21, 5831-7 (1993).

Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Gene77, 51-59 (1989).

Nerlov, C. & Ziff, E. B. EMBO J. 14, 4318-4328 (1995).

Membrane receptors and transporters, including, for example, the insulinreceptor and receptors for certain neurotransmitters, are regulated bythe redox state of the cell. A very large number of enzymes are alsoregulated by the cell's redox state. A partial list of proteins whosefunction is regulated by oxidation-reduction is presented in Table 1.

TABLE 1 Some proteins whose function is regulated by the redox state ofthe cell. References are given within parentheses Enzymes Collagenase(146,147) p21Ras guanine nucleotide-binding protein (148) Proteintyrosine phosphatase (149) p56Lck protein tyrosine kinase (150) Glycogenphosphorylase phosphatase (151) Glycogen synthase (151)Phosphofructokinase (151) Fructose-1,6-bisphosphatase (151) Hexokinase(151) Pyruvate kinase (151,152) Glucose-6-phosphate dehydrogenase (151)3-Hydroxy-3-methylglutaryl CoA reductase (151) SerotoninN-acetyltransferase (151) Guanylate cyclase (151) Medium-chain fattyacyl CoA dehydrogenase (153) Xanthine dehydrogenase (154) ChloroplastNADP-linked glyceraldehyde-3-phosphate dehydrogenase (155) ChloroplastNADP-linked malate dehydrogenase (155) Chloroplast sedoheptulosebisphosphatase (155) Fructose bisphosphatase (155) NADP-malic enzyme(156) 3α-Hydroxysteroid dehydrogenase (157) DsbA protein disulfideisomerase from E. coli (158) Creatine kinase (152) Sarcoplasmicreticulum Ca²⁺-ATPase (152) Transcription factors NF-kappa B (128-130)AP-1 (jun/fos) (131) SoxR (132,133) SoxS (134) OxyR (135)Hypoxia-inducible factor 1 (159) Thyroid transcription factor I (160)Glucocorticoid receptor (161) Sp1 (161,162) Receptors NMDA receptor(163) Insulin receptor NMDA receptor (164,165) Ryanodine receptor (166)HoxB5 (167) c-Myb (167,168) v-Rel (167) p53 (169) Isl-1 (170) OthersErythropoietin RNA-binding protein (171)

These oxidants generally act by effecting alterations in iron-sulfurclusters or by inducing the formation or rupture of disulfide bonds onwhose status the function of the protein depends. B. M. Babior,“Superoxide: a two-edged sword”, Braz J Med Biol Res. February 1997,Volume 30 (2) 141-155. See, also:

Elstner E F (1990). Der Sauerstoff. Biochemie, Biologie, Medizin. B1Wissenschaftsverlag, Mannheim/Wien/Zürich.

Fridovich I (1995). Superoxide radical and superoxide dismutases. AnnualReview of Biochemistry, 64: 97-112.

Halliwell B & Gutteridge J M C (1986). Iron and free radical reactions:two aspects of antioxidant protection. Trends in Biochemical Sciences,11: 372-375.

Bielski B H (1985). Fast kinetic studies of dioxygen-derived species andtheir metal complexes. Philosophical Transactions of the Royal Societyof London, Series B. Biological Sciences, 311: 473-482.

Goldstein S & Czapski G (1986). The role and mechanism of metal ions andtheir complexes in enhancing damage in biological systems or inprotecting these systems from the toxicity of O₂ ⁻ . Free RadicalBiology and Medicine, 2: 3-11.

Harris L R, Cake M H & Macey D J (1994). Iron release from ferritin andits sensitivity to superoxide ions differs among vertebrates.Biochemical Journal, 301: 385-389.

Gardner P R, Rainer I, Epstein L B & White C W (1995). Superoxideradical and iron modulate aconitase activity in mammalian cells. Journalof Biological Chemistry, 270: 13399-13405.

Khan A U & Kasha M (1994). Singlet molecular oxygen in the Haber-Weissreaction. Proceedings of the National Academy of Sciences, USA, 91:12365-12367.

Radi R, Beckman J S, Bush K M & Freeman B A (1991). Peroxynitriteoxidation of sulfhydryls. The cytotoxic potential of superoxide andnitric oxide. Journal of Biological Chemistry, 266: 4244-4250.

Kong S-K, Yim M B, Stadtman E R & Chock P B (1996). Peroxynitritedisables the tyrosine phosphorylation regulatory mechanism:Lymphocyte-specific tyrosine kinase fails to phosphorylate nitratedcdc2(6-20)NH₂ peptide. Proceedings of the National Academy of Sciences,USA, 93: 3377-3382.

Winterbourn C C (1985). Comparative reactivities of various biologicalcompounds with myeloperoxidase-hydrogen peroxide-chloride, andsimilarity of the oxidant to hypochlorite. Biochimica et BiophysicaActa, 840: 204-210.

Thomas E L, Jefferson M M & Grisham M (1982). Myeloperoxidase-catalyzedincorporation of amino acids into proteins: Role of hypochlorous acidand chloramines. Biochemistry, 21: 6299-6308.

Grisham M B, Jefferson M M, Melton D F & Thomas E L (1984). Chlorinationof endogenous amines by isolated neutrophils. Ammonia-dependentbactericidal, cytotoxic, and cytolytic activities of the chloramines.Journal of Biological Chemistry, 259: 10404-10413.

Kanofsky J R, Hoogland H, Wever R & Weiss S J (1988). Singlet oxygenproduction by human eosinophils. Journal of Biological Chemistry, 263:9692-9696.

Steinbeck M J, Khan A U, Karnovsky M J & Hegg G G (1992). Intracellularsinglet oxygen generation by phagocytosing neutrophils in response toparticles coated with a chemical trap. Journal of Biological Chemistry,267: 13425-13433.

McCord J M & Fridovich I (1969). Superoxide dismutase. An enzymicfunction for erythrocuprein. Journal of Biological Chemistry, 244:6049-6055.

Halliwell B & Gutteridge J M C (1989). Free Radicals in Biology andMedicine. 2nd edn. Oxford University Press, Oxford.

Hassan H M & Fridovich I (1996). Enzymatic defenses against the toxicityof oxygen and of streptonigrin in Escherichia coli. Journal ofBacteriology, 129: 1574-1583.

Farr S B, D'Ari R & Touati D (1986). Oxygen-dependent mutagenesis inEscherichia coli lacking superoxide dismutase. Proceedings of theNational Academy of Sciences, USA, 83: 8268-8272.

Ballzan R, Bannister W H, Hunter G J & Bannister J V (1995). Escherichiacoli iron superoxide dismutase targeted to the mitochondria of yeastcells protects the cells against oxidative stress. Proceedings of theNational Academy of Sciences, USA, 92: 4219-4223.

Lapinskas P J, Cunningham K W, Liu X F, Fink G R & Culotta V C (1995).Mutations in PMR1 suppress oxidative damage in yeast cells lackingsuperoxide dismutase. Molecular and Cellular Biology, 15: 1382-1388.

Kelner M J & Bagnell R (1990). Alteration of endogenous glutathioneperoxidase, manganese superoxide dismutase, and glutathione transferaseactivity in cells transfected with a copper-zinc superoxide dismutaseexpression vector: Explanation for variations in paraquat resistance.Journal of Biological Chemistry, 265: 10872-10875.

Yang G, Chan P H, Chen J, Carlson E, Chen S F, Weinstein P, Epstein C J& Kamii H (1994). Human copper-zinc superoxide dismutase transgenic miceare highly resistant to reperfusion injury after focal cerebralischemia. Stroke, 25: 165-170.

Reveillaud I, Phillips J, Duyf B, Hilliker A, Kongpachith A & Fleming JE (1994). Phenotypic rescue by a bovine transgene in a Cu/Zn superoxidedismutase-null mutant of Drosophila melanogaster. Molecular and CellularBiology, 14: 1302-1307.

Imlay J A & Linn S (1988). DNA damage and oxygen radical toxicity.Science, 240: 1302-1309.

Stadtman E R (1992). Protein oxidation and aging. Science, 257:1220-1224.

Thomas C E, Morehouse L A & Aust S D (1985). Ferritin andsuperoxide-dependent lipid peroxidation. Journal of BiologicalChemistry, 260: 3275-3280.

Aikens J & Dix T A (1991). Perhydroxyl radical (HOO.) initiated lipidperoxidation. The role of fatty acid hydroperoxides. Journal ofBiological Chemistry, 266: 15091-15098.

Shigenaga M K, Gimeno C J & Ames B N (1989). Urinary8-hydroxy-2′-deoxyguanosine as a biological marker in in vivo oxidativeDNA damage. Proceedings of the National Academy of Sciences, USA, 86:9697-9701.

Aruoma O I, Halliwell B, Gazewski E & Dizdaroglu M (1989). Damage to thebases in DNA induced by hydrogen peroxide and ferric ion chelates.Journal of Biological Chemistry, 264: 20509-20512.

Demple B & Harrison L (1994). Repair of oxidative damage to DNA:enzymology and biology. Annual Review of Biochemistry, 63: 915-948.

Birnboim H C & Kanabus-Kaminska M (1985). The production of DNA strandbreaks in human leukocytes by superoxide anion may involve a metabolicprocess. Proceedings of the National Academy of Sciences, USA, 82:6820-6824.

Zingarelli B, O'Connor M, Wong H, Salzman A L & Szabó C (1996).Peroxynitrite-mediated DNA strand breakage activates poly-adenosinediphosphate ribosyl synthetase and causes cellular energy depletion inmacrophages stimulated with bacterial lipopolysaccharide. Journal ofImmunology, 156: 350-358.

Burger R M, Projan S J, Horwitz S B & Peisach J (1986). The DNA cleavageof iron-bleomycin. Kinetic resolution of strand scission from basepropenal release. Journal of Biological Chemistry, 261: 15955-15959.

Szabó C, Zingarelli B, O'Connor M & Salzman A L (1996). DNA strandbreakage, activation of poly(ADP-ribose) synthetase, and cellular energydepletion are involved in the cytotoxicity in macrophages and smoothmuscle cells exposed to peroxynitrite. Proceedings of the NationalAcademy of Sciences, USA, 93: 1753-1758.

Stadtman E R & Oliver C N (1991). Metal-catalyzed oxidation of proteins.Physiological consequences. Journal of Biological Chemistry, 266:2005-2008.

Davies K J A, Delsignore M E & Lin S W (1987). Protein damage anddegradation by oxygen radicals. II. Modification of amino acids. Journalof Biological Chemistry, 262: 9902-9907.

Stadtman E R & Berlett B S (1991). Fenton Chemistry. Amino acidoxidation. Journal of Biological Chemistry, 266: 17201-17211.

Oliver C N, Starke-Reed P E, Stadtman E R, Liu G J, Carney J M & Floyd RA (1990). Oxidative damage to brain proteins, loss of glutaminesynthetase activity, and production of free radicals duringischemia/reperfusion-induced injury to gerbil brain. Proceedings of theNational Academy of Sciences, USA, 87: 5144-5147.

Berlett B S, Friguet B, Yim M B, Chock P B & Stadtman E R (1996).Peroxynitrite-mediated nitration of tyrosine residues in Escherichiacoli glutamine synthetase mimics adenylation: Relevance to signaltransduction. Proceedings of the National Academy of Sciences, USA, 93:1776-1780.

Haddad I Y, Pataki G, Calliani C, Beckman J S & Matalon S (1994).Quantitation of nitrotyrosine levels in lung sections of patients andanimals with acute lung injury. Journal of Clinical Investigation, 94:2407-2413.

Albrich J M, McCarthy C A & Hurst J K (1981). Biological reactivity ofhypochlorous acid: Implications for microbicidal mechanisms of leukocytemyeloperoxidase. Proceedings of the National Academy of Sciences, USA,78: 210-214.

Domigan N M, Charlton T S, Duncan M W, Winterburn C C & Kettle A J(1995). Chlorination of tyrosyl residues in peptides by myeloperoxidaseand human neutrophils. Journal of Biological Chemistry, 270:16542-16548.

Bernofsky C, Bandara B M R, Hinojosa O & Strauss S L (1990).Hypochlorite-modified adenine nucleotides: Structure, spin-trapping andformation by activated guinea pig polymorphonuclear leukocytes. FreeRadical Research Communications, 9: 303-315.

Porter N A, Caldwell S E & Mills K A (1995). Mechanisms of free radicaloxidation of unsaturated lipids. Lipids, 30: 277-290.

Halliwell B (1993). The chemistry of free radicals. Toxicology andIndustrial Health, 9: 1-21.

Halliwell B & Chirico S (1993). Lipid peroxidation: Its mechanism,measurement, and significance. American Journal of Clinical Nutrition,57: 715S-725S.

Liu S X, Zhou M, Chen Y, Wen W Y & Sun M J (1996). Lipoperoxidativeinjury to macrophages by oxidatively modified low density lipoproteinmay play an important role in foam cell formation. Atherosclerosis, 121:55-61.

Haberland M E, Fong D & Cheng L (1988). Malondialdehyde-altered proteinoccurs in atheroma of Watanabe heritable hyperlipidemic rabbits.Science, 241: 215-218.

Weitzman S A & Gordon L I (1990). Inflammation and cancer: Role ofphagocyte-generated oxidants in carcinogenesis. Blood, 76: 655-663.

Floyd R A (1990). Role of oxygen free radicals in carcinogenesis andbrain ischemia. FASEB Journal, 4: 2587-2597.

Miesel R, Kurpisz M & Kroger H (1996). Suppression of inflammatoryarthritis by simultaneous inhibition of nitric oxide synthase and NADPHoxidase. Free Radical Biology and Medicine, 20: 75-81.

Adelman R, Saul R L & Ames B N (1988). Oxidative damage to DNA: Relationto species metabolic rate and life span. Proceedings of the NationalAcademy of Sciences, USA, 85: 2706-2708.

Ames B N, Shigenaga M K & Hagen T M (1993). Oxidants, antioxidants. andthe degenerative diseases of aging. Proceedings of the National Academyof Sciences, USA, 90: 7915-7922.

Fridovich I (1975). Superoxide dismutases. Annual Review ofBiochemistry, 44: 147-159.

Fridovich I (1974). Superoxide dismutases. Advances in Enzymology andRelated Areas of Molecular Biology, 41: 35-97.

Hosler B A & Brown Jr R H (1995). Copper/zinc superoxide dismutasemutations and free radical damage in amyotrophic lateral sclerosis.Advances in Neurology, 68: 41-46.

Wiedau-Pazos M, Goto J J, Rabizadeh S, Gralla E B, Roe J A, Lee M K,Valentine J S & Bredesen D E (1996). Altered reactivity of superoxidedismutase in familial amyotrophic lateral sclerosis. Science, 271:515-518.

Deisseroth A & Dounce A L (1970). Catalase: Physical and chemicalproperties, mechanism of catalysis, and physiological role.Physiological Reviews, 50: 319-375.

Michiels C, Raes M, Toussaint O & Remacle J (1994). Importance ofSe-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survivalagainst oxidative stress. Free Radical Biology and Medicine, 17:235-248.

Gaetani G F, Ferraris A M, Rolfo M, Mangerini R, Arena S & Kirkman H N(1996). Predominant role of catalase in the disposal of hydrogenperoxide within human erythrocytes. Blood, 87: 1595-1599.

Cohen H J & Avissar N (1993). Molecular and biochemical aspects ofselenium metabolism and deficiency. Progress in Clinical and BiologicalResearch, 380: 191-202.

Stadtman T C (1990). Selenium biochemistry. Annual Review ofBiochemistry, 59: 111-127.

Burk R F (1990). Protection against free radical injury byselenoenzymes. Pharmacology and Therapeutics, 45: 383-385.

Flohe L (1988). Glutathione peroxidase. Basic Life Sciences, 49:663-668.

Chambers I & Harrison P R (1987). A new puzzle in selenoproteinbiosynthesis: selenocysteine seems to be encoded by the “stop” codon,UGA. Trends in Biochemical Sciences, 12: 255-256.

Cohen H J, Chovaniec M E, Mistretta D & Baker S S (1985). Seleniumrepletion and glutathione peroxidase—differential effects on plasma andred blood cell enzyme activity. American Journal of Clinical Nutrition,41: 735-747.

Anonymous (1980). Treatment of glutathione peroxidase deficiency withvitamin E. Nutrition Reviews, 38: 120-122.

Bigley R, Stankova L, Roos D & Loos J (1980). Glutathione-dependentdehydroascorbate reduction: a determinant of dehydroascorbate uptake byhuman polymorphonuclear leukocytes. Enzyme, 25: 200-204.

Johnson R A, Baker S S, Fallon J T, Maynard III E P, Ruskin J N, Wen Z,Ge K & Cohen H J (1981). An occidental case of cardiomyopathy andselenium deficiency. New England Journal of Medicine, 304 1210-1212.

Anonymous (1980). Prevention of Keshan cardiomyopathy by sodiumselenite. Nutrition Reviews, 38: 278-279.

Maiorino M, Chu F F, Ursini F, Davies K J A, Doroshow J H & Esworthy R S(1991). Phospholipid hydroperoxide glutathione peroxidase is the 18-kDaselenoprotein expressed in human tumor cell lines. Journal of BiologicalChemistry, 266: 7728-7732.

Frei B, England L & Ames B N (1989). Ascorbate is an outstandingantioxidant in human blood plasma. Proceedings of the National Academyof Sciences, USA, 86: 6337-6381.

Meister A (1994). Glutathione-ascorbic acid antioxidant system inanimals. Journal of Biological Chemistry, 269: 9397-9400.

Anonymous (1989). Expanding knowledge of ascorbic acid metabolism.Nutrition Reviews, 47: 360-361.

Levine M (1986). New concepts in the biology and biochemistry ofascorbic acid. New England Journal of Medicine, 314: 892-902.

Chow C K (1991). Vitamin E and oxidative stress. Free Radical Biologyand Medicine, 11: 215-232.

Sies H & Murphy M E (1991). Role of tocopherols in the protection ofbiological systems against oxidative damage. Journal of Photochemistryand Photobiology. B, Biology, 8: 211-218.

Packer L (1991). Protective role of vitamin E in biological systems.American Journal of Clinical Nutrition, 53: 1050S-1055S.

Burton G W & Ingold K U (1989). Vitamin E as an in vitro and in vivoantioxidant. Annals of the New York Academy of Sciences, 570: 7-22.

Koyama K, Takatsuki K & Inoue M (1994). Determination of superoxide andascorbyl radicals in the circulation of animals under oxidative stress.Archives of Biochemistry and Biophysics, 309: 323-328.

Roginsky V A & Stegmann H B (1994). Ascorbyl radical as naturalindicator of oxidative stress: Quantitative regularities. Free RadicalBiology and Medicine, 17: 93-103.

Stankova L, Bigley R, Wyss S R & Aebi H (1979). Catalase anddehydroascorbate reductase in human polymorphonuclear leukocytes (PMN):possible functional relationship. Experientia, 35: 852-853.

Mukai K, Kohno Y & Ishizu K (1988). Kinetic study of the reactionbetween vitamin E radical and alkyl hydroperoxides in solution.Biochemical and Biophysical Research Communications, 155: 1046-1050.

Liebler D C, Kling D S & Reed D J (1986). Antioxidant protection ofphospholipid bilayers by alpha-tocopherol. Control of alpha-tocopherolstatus and lipid peroxidation by ascorbic acid and glutathione. Journalof Biological Chemistry, 261: 12114-12119.

May J M, Qu Z & Morrow J D (1996). Interaction of ascorbate and?-tocopherol in resealed human erythrocyte ghosts. Transmembraneelectron transfer and protection from lipid peroxidation. Journal ofBiological Chemistry, 271: 10577-10582.

Mukai K, Nishimura M, Ishizu K & Kitamura Y (1989). Kinetic study of thereaction of vitamin C with vitamin E radicals (tocopheroxyls) insolution. Biochimica et Biophysica Acta, 991: 276-279.

Weiss S J, Klein R & Slivka A (1982). Chlorination of taurine by humanneutrophils. Journal of Clinical Investigation, 70: 598-607.

Aruoma O I, Halliwell B, Hoey B M & Butler J (1988). The antioxidantaction of taurine, hypotaurine and their metabolic precursors.Biochemical Journal, 256: 251-255.

Wright C E, Lin T T, Lin Y Y, Sturman J A & Gaull G E (1985). Taurinescavenges oxidized chlorine in biological systems. Progress in Clinicaland Biological Research, 179: 137-147.

Weiss S J, Klein R, Slivka A & Wei M (1982). Chlorination of taurine byhuman neutrophils. Evidence for hypochlorous acid generation. Journal ofClinical Investigation, 70: 598-607.

Weiss S J, Lampert M B & Test S T (1983). Long-lived oxidants generatedby human neutrophils: Characterization and bioactivity. Science, 222:625-628.

Babior B M, Kipnes R S & Curnutte J T (1973). Biological defensemechanisms: the production by leukocytes of superoxide, a potentialbactericidal agent. Journal of Clinical Investigation, 52: 741-744.

Curnutte J T & Babior B M (1974). Biological defense mechanisms: theeffect of bacteria and serum on superoxide production by granulocytes.Journal of Clinical Investigation, 53: 1662-1672.

Johnston R B & Newman S L (1977). Chronic granulomatous disease.Pediatric Clinics of North America, 24: 365-376.

Anonymous (1991). A controlled trial of interferon gamma to preventinfection in chronic granulomatous disease. The International ChronicGranulomatous Disease Cooperative Study Group. New England Journal ofMedicine, 324: 509-516.

Chanock S J, El Benna J. Smith R M & Babior B M (1994). The respiratoryburst oxidase. Journal of Biological Chemistry, 269: 24519-24522.

Thomas E L & Fishman M (1986). Oxidation of chloride and thiocyanate byisolated leukocytes. Journal of Biological Chemistry, 261: 9694-9702.

She Z-W, Wewers M D, Herzyk D J, Sagone A L & Davis W B (1989). Tumornecrosis factor primes neutrophils for hypochlorous acid production.American Journal of Physiology, 257: L338-L345.

Raschke P, Becker B F, Leipert B, Schwartz L M, Zahler S & Gerlach E(1993). Postischemic dysfunction of the heart induced by small numbersof neutrophils via formation of hypochlorous acid. Basic Research inCardiology, 88: 321-339.

Harrison J E & Schultz J (1976). Studies on the chlorinating activity ofmyeloperoxidase. Journal of Biological Chemistry, 251: 1371-1374.

Thomas E L, Bozeman P M, Jefferson M M & King C C (1995). Oxidation ofbromide by the human leukocyte enzymes myeloperoxidase and eosinophilperoxidase. Formation of bromamines. Journal of Biological Chemistry,270: 2906-2913.

Weiss S J, Test S T, Eckmann C M, Roos D & Regiani S (1986). Brominatingoxidants generated by human eosinophils. Science, 234: 200-202.

Thomas E L, Grisham M B & Jefferson M M (1983).Myeloperoxidase-dependent effect of amines on functions of isolatedneutrophils. Journal of Clinical Investigation, 72: 441-454.

Rosen H, Orman J, Rakita R M, Michel B R & VanDevanter D R (1990). Lossof DNA-membrane interactions and cessation of DNA synthesis inmyeloperoxidase-treated Escherichia coli. Proceedings of the NationalAcademy of Sciences, USA, 87: 10048-10052.

Larrocha C, deCastro M F, Fontan G, Viloria A, Ferrandoz Chacon J L &Jimenez C (1982). Hereditary myeloperoxidase deficiency: a study of 12cases. Scandinavian Journal of Haematology, 29: 389-397.

Parry M F, Root R K, Metcalf J A, Delaney K K, Kaplow L S & Richar W J(1981). Myeloperoxidase deficiency: Prevalence and clinicalsignificance. Annals of Internal Medicine, 95: 293-301.

Omar B A, Gad N M, Jordan M C, Striplin S P, Russell W J, Downey J M &McCord J M (1990). Cardioprotection by Cu,Zn-superoxide dismutase islost at high doses in the reoxygenated heart. Free Radical Biology andMedicine, 9: 465-471.

Omar B A & McCord J M (1990). The cardioprotective effect ofMn-superoxide dismutase is lost at high doses in the postischemicisolated rabbit heart. Free Radical Biology and Medicine, 9: 473-478.

Scott M D, Meshnick S R & Eaton J W (1989). Superoxide dismutaseamplifies organismal sensitivity to ionizing radiation. Journal ofBiological Chemistry, 264: 2498-2501.

Scott M D, Meshnick S R & Eaton J W (1987). Superoxide dismutase-richbacteria. Paradoxical increase in oxidant toxicity. Journal ofBiological Chemistry, 262: 3640-3645.

Winterbourn C C (1981). Cytochrome c reduction by semiquinone radicalscan be indirectly inhibited by superoxide dismutase. Archives ofBiochemistry and Biophysics, 209: 159-167.

Cadenas E (1989). Biochemistry of oxygen toxicity. Annual Review ofBiochemistry, 58: 79-110.

Forage R G & Foster M A (1979). Resolution of the coenzymeB-12-dependent dehydratases of Klebsiella sp. and Citrobacter freundii.Biochimica et Biophysica Acta, 569: 249-258.

Meier B, Cross A R, Hancock J T, Kaup F J & Jones O T G (1991).Identification of a superoxide-generating NADPH oxidase system in humanfibroblasts. Biochemical Journal, 275: 241-245.

Meier B, Radeke H H, Selle S, Habermehl G G, Resch K & Sies H (1990).Human fibroblasts release low amounts of reactive oxygen species inresponse to the potent phagocyte stimulants, serum-treated zymosan,N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4 or12-O-tetradecanoylphorbol 13-acetate. Biological Chemistry Hoppe-Seyler,371: 1021-1025.

Meier B, Radeke H H, Selle S, Younes M, Sies H, Resch K & Habermehl G G(1989). Human fibroblasts release reactive oxygen species in response tointerleukin-1 or tumour necrosis factor-α. Biochemical Journal, 263:539-545.

Schreck R, Meier B, Mannel D N, Droge W & Baeuerle P A (1992).Dithiocarbamates as potent inhibitors of nuclear factor kappa Bactivation in intact cells. Journal of Experimental Medicine, 175:1181-1194.

Griendling K K, Minieri C A, Ollerenshaw J D & Alexander R W (1994).Angiotensin II stimulates NADH and NADPH oxidase activity in culturedvascular smooth muscle cells. Circulation Research, 74: 1141-1148.

Rajagopalan S, Surz S, Munzel T, Tarpey M, Freeman B A, Griendling K K &Harrison D G (1996). Angiotensin II-mediated hypertension in the ratincreases vascular superoxide production via membrane NADH/NADPH oxidaseactivation. Contribution to alterations of vasomotor tone. Journal ofClinical Investigation, 97: 1916-1923.

Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Y & Kimura H(1995). Amyloid peptides are toxic via a common oxidative mechanism.Proceedings of the National Academy of Sciences, USA, 92: 1989-1993.

Heinecke J W & Shapiro B M (1989). Respiratory burst oxidase offertilization. Proceedings of the National Academy of Sciences, USA, 86:1259-1263.

Aitken R J & Clarkson J S (1987). Cellular basis of defective spermfunction and its association with the genesis of reactive oxygen speciesby human spermatozoa. Journal of Reproduction and Fertility, 81:459-469.

De Lamirande E, Eiley D & Gagnon C (1993). Inverse relationship betweenthe induction of human sperm capacitation and spontaneous acrosomereaction by various biological fluids and the superoxide scavengingcapacity of these fluids. International Journal of Andrology, 16:258-266.

Acker H, Bolling B, Delpiano M A, Dufau E, Gorlach A & Holtermann G(1992). The meaning of H₂O₂ generation in carotid body cells for pO₂chemoreception. Journal of the Autonomic Nervous System, 41: 41-51.

Cross A R, Henderson L, Jones O T, Delpiano M A, Hentschel J & Acker H(1990). Involvement of an NAD(P)H oxidase as a pO₂ sensor protein in therat carotid body. Biochemical Journal, 272: 743-747.

Kummer W & Acker H (1995). Immunohistochemical demonstration of foursubunits of neutrophil NAD(P)H oxidase in type I cells of carotid body.Journal of Applied Physiology, 78: 1904-1909.

Schreck R, Rieber P & Baeuerle P A (1991). Reactive oxygen intermediatesas apparently widely used messengers in the activation of the NF-κBtranscription factor and HIV-1. EMBO Journal, 10: 2247-2258.

Menon S D, Quin S, Guy G R & Tan Y H (1993). Differential induction ofnuclear NF-κB by protein phosphatase inhibitors in primary andtransformed human cells. Requirement for both oxidation andphosphorylation in nuclear translocation. Journal of BiologicalChemistry, 268: 26805-26812.

Baeuerle P A & Henkel T (1994). Function and activation of NF-κB in theimmune system. Annual Review of Immunology, 12: 141-179.

Puri P L, Avantaggiati M L, Burgio V L, Chirillo P, Collepardo D, NatoliG, Balsano C & Levrero M (1995). Reactive oxygen intermediates mediateangiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity andproliferative hypertrophic responses in myogenic cells. Journal ofBiological Chemistry, 270: 22129-22134.

Park S J & Gunsalus R P (1995). Oxygen, iron, carbon, and superoxidecontrol of the fumarase fumA and fumC genes of Escherichia coli: Role ofthe arcA ,fnr, and soxR gene products. Journal of Bacteriology, 177:6255-6262.

Hidalgo E & Demple B (1996). Activation of SoxR-dependent transcriptionin vitro by noncatalytic or NifS-mediated assembly of [2Fe-2S] clustersin Apo-SoxR. Journal of Biological Chemistry, 271: 7269-7272.

Jair K W, Fawcett W P, Fujita N, Ishihama A & Wolf Jr R E (1996).Ambidextrous transcriptional activation by SoxS: Requirement for theC-terminal domain of the RNA polymerase alpha subunit in a subset ofEscherichia coli superoxide-inducible genes. Molecular Microbiology, 19:307-317.

Christman M F, Storz G & Ames B N (1989). OxyR, a positive regulator ofhydrogen peroxide-inducible genes in Escherichia coli and Salmonellatyphimurium, is homologous to a family of bacterial regulatory proteins.Proceedings of the National Academy of Sciences, USA, 86: 3484-3488.

Marin-Hincapie M & Garofalo R S (1995). Drosophila insulin receptor:lectin-binding properties and a role for oxidative-reduction of receptorthiols in activation. Endocrinology, 136: 2357-2366.

Pan Z H, Bahring R, Grantyn R & Lipton S A (1995). Differentialmodulation by sulfhydryl redox agents and glutathione of GABA- andglycine-evoked currents in rat retinal ganglion cells. Journal ofNeuroscience, 15: 1384-1391.

Staal F J T, Anderson M T, Staal G E J, Herzenberg L A & Gitler C(1994). Redox regulation of signal transduction: Tyrosinephosphorylation and calcium influx. Proceedings of the National Academyof Sciences, USA, 91: 3619-3622.

Hidalgo E, Bollinger Jr J M, Bradley T M, Walsh C T & Demple B (1995).Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein androle of the metal centers in transcription. Journal of BiologicalChemistry, 270: 20908-20914.

Flint D H, Tuminello J F & Emptage M H (1993). The inactivation of Fe-Scluster containing hydrolases by superoxide. Journal of BiologicalChemistry, 268: 22369-22376.

Halliwell B (1992). Switches in enzymes. Nature, 354: 191-192.

Bandyopadhyay S & Gronostajski R M (1994). Identification of a conservedoxidation-sensitive cysteine residue in the NF1 family of DNA-bindingproteins. Journal of Biological Chemistry, 269: 29949-29955.

Landgraf W, Regulla S, Meyer H E & Hofmann F (1991). Oxidation ofcysteines activates cGMP-dependent protein kinase. Journal of BiologicalChemistry, 266: 16305-16311.

Hayashi T. Ueno Y & Okamoto T (1993). Oxidoreductive regulation ofnuclear factor kappa B: Involvement of a cellular reducing catalystthioredoxin. Journal of Biological Chemistry, 268: 11380-11388.

Petronilli V, Constantini P, Scorrano L. Colonna R, Passamonti S &Bernardi P (1994). The voltage sensor of the mitochondrial permeabilitytransition pore is tuned by the oxidation-reduction state of vicinalthiols. Journal of Biological Chemistry, 269: 16638-16642.

Weiss S J, Peppin G, Ortiz X, Ragsdale C & Test S T (1985). Oxidativeautoactivation of latent collagenase by human neutrophils. Science, 227:747-749.

Weiss S J & Peppin G J (1986). Collagenolytic metalloenzymes of thehuman neutrophil. Characteristics, regulation and potential function invivo. Biochemical Pharmacology, 35: 3189-3197.

Lander H M, Ogiste J S, Teng K K & Novogrodsky A (1995). p21ras as acommon signaling target of reactive free radicals and cellular redoxstress. Journal of Biological Chemistry, 270: 21195-21198.

Fialkow L, Chan C K, Grinstein S & Downey G P (1993). Regulation oftyrosine phosphorylation in neutrophils by the NADPH oxidase. Role ofreactive oxygen intermediates. Journal of Biological Chemistry, 268:17131-17137.

Hardwick J S & Sefton M B (1995). Activation of the Lck tyrosine proteinkinase by hydrogen peroxide requires the phosphorylation of Tyr-394.Proceedings of the National Academy of Sciences, USA, 92: 4527-4531.

Ziegler D M (1985). Role of reversible oxidation-reduction of enzymethiols-disulfides in metabolic regulation. Annual Review ofBiochemistry, 54: 305-329.

Korge P & Campbell K B (1993). The effect of changes in iron redox stateon the activity of enzymes sensitive to modification of SH groups.Archives of Biochemistry and Biophysics, 304: 420-428.

Johnson B D, Mancini-Samuelson G J & Stankovich M T (1995). Effect oftransition-state analogues on the redox properties of medium-chainacyl-CoA dehydrogenase. Biochemistry, 34: 7047-7055.

Hassoun P M, Yu F S, Zulueta J J, White A C & Lanzillo J J (1995).Effect of nitric oxide and cell redox status on the regulation ofendothelial cell xanthine dehydrogenase. American Journal of Physiology,268: L809-L817.

Li D, Stevens F J, Schiffer M & Anderson L E (1994). Mechanism of lightmodulation: Identification of potential redox-sensitive cysteines distalto catalytic site in light-activated chloroplast enzymes. BiophysicalJournal, 67: 29-35.

Drincovich M F & Andreo C S (1994). Redox regulation of maize NADP-malicenzyme by thiol-disulfide interchange: effect of reduced thioredoxin onactivity. Biochimica et Biophysica Acta, 1206: 10-16.

Terada T, Nanjo H, Shinagawa K, Umemura T, Nishinaka T, Mizoguchi T &Nishihara T (1993). Modulation of 3 ?-hydroxysteroid dehydrogenaseactivity by the redox state of glutathione. Journal of EnzymeInhibition, 7: 33-41.

Wunderlich M, Jaenicke R & Glockshuber R (1993). The redox properties ofprotein disulfide isomerase (DsbA) of Escherichia coli result from atense conformation of its oxidized form. Journal of Molecular Biology,233: 559-566.

Wang G L, Jiang B H & Semenza G L (1995). Effect of altered redox stateson expression and DNA-binding activity of hypoxia-inducible factor 1.Biochemical and Biophysical Research Communications, 212: 550-556.

Arnone M I, Zannini M & Di Lauro R (1995). The DNA binding activity andthe dimerization ability of the thyroid transcription factor I are redoxregulated. Journal of Biological Chemistry, 270: 12048-12055.

Esposito F, Cuccovillo F, Morra F, Russo T & Cimino F (1995). DNAbinding activity of the glucocorticoid receptor is sensitive to redoxchanges in intact cells. Biochimica et Biophysica Acta, 1260: 308-314.

Ammendola R, Mesuraca M, Russo T & Cimino F (1994). The DNA-bindingefficiency of Sp1 is affected by redox changes. European Journal ofBiochemistry, 225: 483-489.

Gozlan H, Khazipov R & Ben-Ari Y (1995). Multiple forms of long-termpotentiation and multiple regulatory sites of N-methyl-D-aspartatereceptors: role of the redox site. Journal of Neurobiology, 26: 360-369.

Sullivan J M, Traynelis S F, Chen H S, Escobar W, Heinemann S F & LiptonS A (1994). Identification of two cysteine residues that are requiredfor redox modulation of NMDA subtype of glutamate receptor. Neuron, 13:929-936.

Tang L H & Aizenman E (1993). Long-lasting modification of theN-methyl-D-aspartate receptor channel by a voltage-dependent sulfhydrylredox process. Molecular Pharmacology, 44: 473-478.

Liu G & Pessah I N (1994). Molecular interaction between ryanodinereceptor and glycoprotein triadin involves redox cycling of functionallyimportant hyperreactive sulfhydryls. Journal of Biological Chemistry,269: 33028-33034.

Galang C K & Hauser C A (1993). Cooperative DNA binding of the humanHoxB5 (Hox-2.1) protein is under redox regulation in vitro. Molecularand Cellular Biology, 13: 4609-4617.

Myrset A H, Bostad A, Jamin N, Lirsac P N, Toma F & Gabrielsen O S(1993). DNA and redox state induced conformational changes in theDNA-binding domain of the Myb oncoprotein. EMBO Journal, 12: 4625-4633.

Hainaut P & Milner J (1993). Redox modulation of p53 conformation andsequence-specific DNA binding in vitro. Cancer Research. 53: 4469-4473.

Sanchez-Garcia I & Rabbitts T H (1993). Redox regulation of in vitroDNA-binding activity by the homeodomain of the Isl-1 protein. Journal ofMolecular Biology, 231: 945-949.

Rondon I J, Scandurro A B, Wilson R B & Beckman B S (1995). Changes inredox affect the activity of erythropoietin RNA binding protein. FEBSLetters, 359: 267-270.

Michael Story and Reinhard Kodym, Signal Transduction During Apoptosis;Implications For Cancer Therapy, Frontiers in Bioscience, 3, d365-375,(Mar. 23, 1998).

Reactive oxygen species (ROS) are implicated in the pathogenesis of awide variety of human diseases. Recent evidence suggests that atmoderately high concentrations, certain forms of ROS such as H₂O₂ mayact as signal transduction messengers. At least two well-definedtranscription factors, nuclear factor (NF-κB) and activator protein(AP)-1 have been identified to be regulated by the intracellular redoxstate. R. Schreck, P. Rieber & P. A. Baeuerle, Reactive oxygenintermediates as apparently widely used messengers in the activation ofthe NF-kappa B transcription factor and HIV-1. EMBO J 10: 2247-2258(1991). Binding sires of the redox-regulated transcription factors NF-κBand AP-1 are located in the promoter region of a large variety of genesthat are directly involved in the pathogenesis of diseases, e.g.. AIDS,cancer, atherosclerosis and diabetic complications. Biochemical andclinical studies have indicated that antioxidant therapy may be usefulin the treatment of disease. Critical steps in the signal transductioncascade are sensitive to oxidants and antioxidants. Many basic events ofcell regulation such as protein phosphorylation and binding oftranscription factors to consensus sites on DNA are driven byphysiological oxidant-antioxidant homeostasis, especially by thethiol-disulfide balance. Endogenous glutathione and thioredoxin systemsmay therefore be considered to be effective regulators ofredox-sensitive gene expression. By controlling redox cascades by usingantioxidants, for example, treatments for several diseases may bepossible, such as hemotogenic cancer cell metastasis and AIDS. Sen, C.K., Packer, L. Antioxidant and redox regulation of gene transcription.FASEB J. 10, 709-720 (1996). See, also:

Packer L, Roy S, Sen C K, a-Lipoic acid: a metabolic antioxidant andpotential redox modulator of transcription Advances in Pharmacology1996; 38: 79-101.

Sen, C. K., S. Roy, and L. Packer. Therapeutic potential of theantioxidant and redox properties of alpha-lipoic acid. In: OxidativeStress, Cancer, AIDS and Neurodegenerative Diseases. Eds. L. Montagnier,R. Olivier, C. Pasquier. Marcel Dekker Inc., New York, in press.

Packer L, Witt E H, Tritschler H J. Alpha-lipoic acid as a biologicalantioxidant. Free Rad. Biol. Med. 1995; 19: 227-250.

Sen C K, Atalay M, Hanninen O. Exercise-induced oxidative stress:glutathione supplementation and deficiency. J. Appl. Physiol. 1994; 77:2177-2187.

Roy S, Sen C K, Tritschler H J, Packer L. Modulation of cellularreducing equivalent homeostasis by a-lipoic acid: mechanisms andimplications for diabetes and ischemic injury. Biochem. Pharmacol., inpress, 1996.

Arne E S J, Nordberg J, Holmgren A. Efficient reduction of lipoamide andlipoic acid by mammalian thiredoxin reductase. Biochem. Biophys. Res.Commun. 1996 in press.

Rosenberg H R, Culik R. Effect of a-lipoic acid on vitamin C and vitaminE deficiencies. Arch. Biochem. Biophys 1959; 80: 86-93.

Baeuerle P A, Henkel T. Function and activation of NF-kB in immunesystem. Annu. Rev. Immunol. 1994; 12: 141-179.

Staal F J T, Roederer M, Herzenberg L A, Herzenberg L A. Intracellularthiols regulate activation of nuclear factor kappa B and transcriptionof human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 1990; 87:9943-9947.

Sen C K, Roy S, Packer L. Involvement of intracellular Ca2+ inoxidant-induced NF-kB activation. FEBS Letters 1996; 385: 58-62.

Watt F, Molloy P L. Specific cleavage of transcription factors by thiolprotease, m-calpain. Nucleic Acid Res. 1993; 21: 5092-5100.

Yan C H I, Ferrari G, Greene L A. N-Acetylcysteine-promoted survival ofPC12 cells is glutathione-independent but transcription-dependent. J.Biol. Chem. 1995; 270: 26827-26832

Baur A, Harrer T, Peukert M, Jahn G, Kalden J R, Fleckenstein B.Alpha-lipoic acid is an effective inhibitor of human immuno-deficiencyvirus (HIV-1) replication. Klin. Wochenschr. 1991; 69: 722-724.

Papp B, Bryn R A. Stimulation of HIV expression by intracellular calciumpump inhibition. J. Biol. Chem. 1995; 270: 10275-10283.

Eck H P, Gmunder H, Hartmann M, Petzoldt D, Daniel V, Droge W. Lowconcentrations of acid soluble thiol (cysteine) in blood plasma of HIV-1infected patients. Biol. Chem. Hoppe-Seyler 1989; 370: 101-108.

Droege W, Eck H-P, Naher H, Pekar U, Daniel V. Abnormal amino-acidconcentrations in blood of patients with acquired immunodeficiencysyndrome (AIDS) may contribute to the immunological defect. Biol. Chem.Hoppe. Seyler 1988; 369: 143-148.

Droege W, Eck H-P, Mihm S. HIV-induced cysteine deficiency and T celldysfunction—a rationale for treatment with N-acetylcysteine. Immunol.Today. 1992; 13: 211-214.

Roederer M, Staal F J T, Anderson M E, Rabin R, Raju P A, Herzenberg L.A, Herzenberg L A. Disregulation of leukocyte glutathione in AIDS. Ann.NY Acad. Sci. 1993: 677: 113-125. 20.Herzenberg L et al. In: OxidativeStress, Cancer, AIDS and Neurodegenerative Diseases. Eds. L. Montagnier,R. Olivier, C. Pasquier, Marcel Dekker Inc., New York, in press.

Merin J P, Matsuyama M, Kira T, Baba M, Okamoto T. a-Lipoic acid blocksHIV-1 LTR-dependent expression of hygromycin resistance in THP-1 stabletransforms. FEBS Letters; 1996, in press.

The heat-shock (HS) response is a ubiquitous cellular response tostress, involving the transcriptional activation of HS genes. H₂O₂ hasbeen shown to induce a concentration-dependent transactivation andDNA-binding activity of heat-shock factor-1 (HSF-1). DNA-bindingactivity was, however, lower with H₂O₂ than with HS, thus providingevidence of a dual regulation of HSF by oxidants. The effects of H₂O₂ invitro were reversed by the sulphydryl reducing agent dithiothreitol andthe endogenous reductor thioredoxin (TRX). In addition, TRX alsorestored the DNA-binding activity of HSF oxidized in vivo, while it wasfound to be itself induced in vivo by both HS and H₂O₂. Thus, H₂O₂exerts dual effects on the activation and the DNA-binding activity ofHSF: on the one hand, H₂O₂ favours the nuclear translocation of HSF,while on the other, it alters HSF-DNA-binding activity, most likely byoxidizing critical cysteine residues within the DNA-binding domain. HSFthus belongs to the group of ROS-modulated transcription factors. MurielR. Jacquier-Sarlin and Barbara S. Polla, “Dual regulation of heat-shocktranscription factor (HSF) activation and DNA-binding activity by H₂O₂:role of thioredoxin”. (1996)

The mammalian stress response evokes a series of neuroendocrineresponses that activate the hypothalamic-pituitary-adrenal (HPA) axisand the sympathetic nervous system. Coordinated interactions betweenstress response systems, occurring at multiple levels including thebrain, pituitary gland, adrenal gland, and peripheral tissues, arerequired for the maintenance of homeostatic plateau. Glucocorticoids, asa major peripheral effector of the HPA axis, play an essential role inre-establishing homeostatic status in every peripheral tissue in human.On the other hand, the adaptive responses are also operated againstvarious intrinsic or extrinsic forces that disturb cellular homeostasisas a part of local host-defense mechanisms at a cellular level.Currently, reduction/oxidation (redox) reactions are intimately involvedin the control of biological processes including modulation of thefunction of transcription factors, e.g., AP-1 and NF-κB. Cells containendogenous buffering systems against excessive production of reactiveoxygen intermediates (ROIs) to preserve cellular metabolism through theexpression and regulation of many enzymes.

Glucocorticoids, on binding to the glucocorticoid receptor (GR), promotethe dissociation of heat shock proteins (HSPs), and the ligand-receptorcomplex translocates to the nucleus then binds to palindromic DNAsequences, called glucocorticoid response elements (GREs). After bindingto DNA, the GR differentially regulates target gene expression toproduce hormone action, interacting with or without other transcriptionfactors and coactivators/corepressors. The GR has a modular structuremainly consisting of a central DNA binding domain (DBD), nuclearlocalization signals, a ligand binding domain (LBD), and severaltranscription activation functions. The human GR contains 20 cysteineresidues, concentrated in the central region spanning the DBD and LBD.The cysteine residues in each domain have been shown to be crucial formaintaining both structure and function of those domains. For examples,it has already been shown that conversion of sulfhydryls in the DBD todisulfides blocks GR binding to DNA cellulose, and that metal ions whichhave high affinity for thiols interfere with the DBD-DNA interaction.

The TRX system operates as an endogenous defense machinery forglucocorticoid-mediated stress responses against oxidative stress. TRXis considered to be involved in transcriptional processes: for example,NF-κB activation is inhibited, whereas AP-1 activity is induced by TRX.Moreover, the GR in the isolated rat cytosol is shown to be stabilizedand maintained in their reduced, ligand-binding form by TRX. Thefunctional interaction between cellular oxistress, TRX, and GR, andindicate that cellular redox state and TRX levels are importantdeterminants of cellular sensitivity to glucocorticoids. Thus, TRXsystems may control homeostasis not only by, for example, sequestratingROIs, but also by fine tuning of hormonal signals. These phenomenaappear to be rationale, for example during inflammation, where cells arebelieved to be exposed to severe oxidative stress, where suppression ofglucocorticoid action may potentiate endogenous defense mechanisms andprevent premature termination of the cascade of inflammatory reactionsfor self defense. Increase in cellular TRX levels may restore thereceptor activity and permit the GR to efficiently communicate withtarget genes. Resultant activation of anti-inflammatory genes and/orrepression of inflammatory genes may prevent overshoot of inflammation.This process may be modulated by an alteration of the redox potential ofthe cell and the concentration of reduced GSH in the intracellularfluid. Yuichi Makino, Kensaku Okamoto, Kiichi Hirota, Junji Yodoi,Kazuhiko Umesono, Isao Makino, and Hirotoshi Tanaka, Cross-Talk betweenEndocrine Control of Stress Response and Cellular Antioxidant DefenseSystem, Thioredoxin is a Redox-Regulating Cellular Cofactor forGlucocorticoid Hormone Action (Poster), Proceedings of 3rd InternetWorld Congress on Biomedical Sciences, 1996.12.9-20 Riken, Tsukuba,Japan. Therefore, glucocorticoid function may be modulated byglutathione administration. Thus, treatment of chronic inflammatoryconditions, such as rheumatoid arthritis, as well as other immune andautoimmune disorders, may also benefit from treatment with glutathione.See:

Kuehl, F. A., Ham, E. A., Egan, R. W., Dougherty, H. W., Bonney, R. J.and Humes, J. L.: Studies on a destructive oxidant released in theenzymatic reduction of prostaglandin G2 and other hydroperoxy acids. In:Pathology of Oxygen, ed. A. P. Auton, Acad. Press, New York, 1982, pp.175-190.

Lash, L. H., Hagen, T. M., & Jones, D. P.: Exogenous glutathioneprotects intestinal epithelial cells from oxidative injury. Proc. Natl.Acad. Sci. USA 83: 4641-4645, 1986.

Selye, H. 1946. The general adaptation syndrome and the diseases ofadaptation. J. Clin. Endocrinol. Metab. 6: 117-230.

Munck, A., P. M. Guyre, and N. J. Holbrook. 1984. Physiologicalfunctions of glucocorticoids in stress and their relation topharmacological actions. Endocrine Rev. 5: 25-44.

Yu, B. P. 1994. Cellular defenses against damage from reactive oxygenspecies. Physiol. Rev. 74: 139-162.

Bauskin, A. R., I. Aikalay, and Y. Ben-Neriah. 1991. Redox regulation ofa protein tyrosine kinase in the endoplasmic reticulum. Cell 66:685-696.

Demple, B., and C. F. Amabile-Cuevas. 1991. Redox redux: the control ofoxidative stress responses. Cell 67: 837-839.

Firth, J. D., B. L. Ebert, C. W. Pugh, and P. J. Ratcliffe. 1994.Oxygen-regulated control elements in the phosphoglycerate kinase andlactate dehydrogenase A genes: similarities with the erythropoietin 3′enhancer. Proc. Natl. Acad. Sci. USA. 91: 6496-6500.

Devary, Y., R. A. Gottlieb, T. Smeal, and M. Karin. 1992. The mammalianultraviolet is triggered by activation of Src tyrosine kinases. Cell 71:1081-1091.

Schreck, R, P. Rieber, and P. A. Baeuerle. 1991. Reactive oxygenintermediates as apparently widely used messengers in the activation ofthe NF-kB transcription factor and HIV-1. EMBO J. 10: 2247-2258.

Abate, C., L. Patel, F. J. Rauscher, III, and T. Curran. 1990. Redoxregulation of Fos and Jun DNA-binding activity in vitro. Science 249:1157-1161.

Klebanoff, S. J., M. A. Vadas, J. M. Harlan, L. H. Sparks, J. R. Gamble,J. M. Agosti, and A. M. Waltersdorf. 1986. Stimulation of neutrophils bytumor necrosis factor. J. Immunol. 136, 4220-4225.

Yoshie, O., T. Majima, and H. Saito. 1989. Membrane oxidative metabolismof human eosinophilic cell line EoL-1 in response to phorbol diester andformyl peptide: synergistic augmentation by interferon-gamma and tumornecrosis factor. J. Leukocyte Biol. 45, 10-20.

DeChatelet, L. R., P. S. Shirley, and R. B. Johnston. 1976. Effect ofphorbol myristate acetate on the oxidative metabolism of humanpolymorphonuclear leukocytes. Blood 47, 545-554.

Beato M, P. Herrlich, and G. Schütz. 1995. Steroid receptors: many actors in search of a plot. Cell 83: 851-857.

Evans, R. M. 1988. The steroid and thyroid hormone receptor superfamily.Science 240: 889-895.

Glass, K. C. 1994. Differential recognition of target genes by nuclearreceptor monomers, dimers, and heterodimers. Mol. Endocrinol. 15:391-407.

Hörlein, A. J., A. M. N{umlaut over (aa)}r, T. Heinzel, J. Torchis, B.Gloss, R. Kurokawa, A. Ryan, Y. Kamei, M. Söderström, C. K. Glass, andM. G. Rosenfeld. 1995. Ligand-independent repression by the thyroidhormone receptor mediated by a nuclear receptor co-repressor. Nature377: 397-404.

Katzenellenbogen, J. A., B. W. O'Malley, and B. S. Katzellenbogen. 1996.Tripartite steroid hormone receptor pharmacology: interaction withmultiple effector sites as a basis for the cell- and promoter-specificaction of these hormones. Mol. Endocrinol. 10: 119-131.

Onate, S. A., S. Y. Tsai, M.-J., Tsai, and B. W. O'Malley. 1995.Sequence and characterization of a coactivator for the steroid hormonereceptor superfamily. Science 270: 1354-1357.

Kamei, Y., L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.-C.Lin, R. A. Heyman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1996. ACBP integrator complex mediates transcriptional activation and AP-1inhibition by nuclear receptors. Cell 85: 403-414.

Chakraborti, P. K., M. J. Garabedian, K. R. Yamamoto, and S. S. Simons,Jr. 1992. Role of cysteines 640, 656, and 661 in steroid binding to ratglucocorticoid. J. Biol. Chem. 267: 11366-11373.

Simons, S. S. Jr, and W. B. Pratt. 1995. Glucocorticoid receptor thiolsand steroid-binding activity. Methods Enzymol. 251: 406-422.

Luisi, B. F., W. X. Xu, Z. Otwinowski, L. P. Freedman, K. R. Yamamoto,and P. B. Sigler. 1991. Crystallographic analysis of the interaction ofthe glucocorticoid receptor with DNA. Nature 352: 497-505.

Bodwell, J. E., N. J. Holbrook, and A. Munck. 1984. Sulfhydryl-modifyingreagents reversibly inhibit binding of glucocorticoid-receptor complexesto DNA-cellulose. Biochemistry 23: 1392-1398.

Makino Y., H. Tanaka, K. Dahlman-Wright, and I. Makino. 1996. Modulationof glucocorticoid-inducible gene expression by metal ions. Mol.Pharmacol. 49: 612-620.

Holmgren, A. 1995. Thioredoxin structure and mechanism: conformationalchanges on oxidation of the active-site sulfhydryls to a disulfide.Structure 3: 239-243. 26. Holmgren, A. 1985. Thioredoxin. Annu. Rev.Biochem. 54: 237-271.

Tagaya, Y., Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro, N.Brown, K.-I. Arai, T. Yokota, H. Wakasugi, and J. Yodoi. 1989.ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous tothioredoxin; possible involvement of dithiol-reduction in the IL-2receptor induction. EMBO J. 8: 757-764.

Tagaya, Y., M. Okada, K. Sugie, T. Kasahara, N. Kondo, J. Hamuro, K.Matsushima, C. A. Dinarello, and J. Yodoi. 1988. IL-2 receptor(p55)Tac-inducing factor. Purification and characterization of adult Tcell leukemia-derived factor. J. Immunol. 140: 2614-2620.

Wakasugi, N., Y. Tagaya, H. Wakasugi, A. Mitsui, M. Maeda, J. Yodoi, andT. Tursz. 1990. Adult T-cell leukemia-derived factor/tyhioredoxin,produced by both human T-lymphotrophic virus type I and Epstein-Barrvirus-transformed lymphocytes, acts as an autocrine growth factor andsynergizes with interleukin 1 and interleukin 2. Proc. Natl. Acad. Sci.USA. 87: 8282-8286.

Schenk, H., M. Klein, W. Erdbrugger, W. Droge, and K. Schulze-Osthoff.1994. Distinct effects of thioredoxin and antioxidants on the activationof transcription factors NF-kB and AP-1. Proc. Natl. Acad. Sci. USA. 91:1672-1676.

Meyer, M., R. Schreck, and P. A. Baeuerle. 1993. H2O2 and antioxidantshave opposite effects on activation of NF-kB and AP-1 in intact cells:AP-1 as secondary antioxidant-responsive factor. EMBO J. 12: 2005-2015.

Grippo, J. F., A. Holmgren, and W. B. Pratt. 1985. Proof that theendogenous, heat-stable glucocorticoid receptor-activating factor isthioredoxin. J. Biol. Chem. 260: 93-97.

Makino, Y., K. Okamoto, N. Yoshikawa, M. Aoshima, K. Hirota, J. Yodoi,K. Umesono, I. Makino, and H. Tanaka. Thioredoxin: a Redox-RegulatingCellular Cofactor for Glucocorticoid Hormone Action. J. Clin. Invest.(in press)

Sasada, T., S. Iwata, N. Sato, Y. Kitaoka, K. Hirota, K. Nakamura, A.Nishiyama, Y. Taniguchi, A. Takabayashi, and J. Yodoi. 1996. Redoxcontrol of resistence to cis-diamminedichloroplatinum (II) (CDDP).Protective effect of human thioredoxin against CDDP-inducedcytotoxicity. J. Clin. Invest. 97: 2268-2276.

Alksnis, M., T. Barkhem, P.-E. Strömstedt, H. Ahola, E. Kutoh, J.-Å.Gustafsson, L. Poellinger, and S. Nilson. 1991. High level expression offunctional full length and truncated glucocorticoid receptor in Chinesehamster ovary cells. J. Biol. Chem. 266: 10078-10085.

Tagaya, Y., H. Wakasugi, H. Masutani, H. Nakamura, S. Iwata, A. Mitsui,S. Fujii, N. Wakasugi, T. Tursz, and J. Yodoi. 1990. Role of ATL-derivedfactor (ADF) in the normal and abnormal cellular activation: involvementof dithiol related reduction. Mol. Immunol. 27: 1279-1289.

Rangarajan, P. N., K. Umesono, and R. M. Evans. 1992. Modulation ofglucocorticoid receptor function by protein kinase A. Mol. Endocrinol.6: 1451-1457.

Matthews, J. R., N. Wakasugi, J.-L. Virelizer, J. Yodoi, and R. T. Hay.1992. Thioredoxin regulates the DNA binding activity of NF-kB byreduction of a disulphide bond involving cysteine 62. Nucleic Acids Res.20: 3821-3830.

Yokomizo, A., M. Ono, H. Nanri, Y. Makino, T. Ohga, M. Wada, T. Okamoto,J. Yodoi, M. Kuwano, and K. Kohno. 1995. Cellular levels of thioredoxinassociated with drug sensitivity to cisplatin, mitomycin C, doxorubicin,and etoposide. Cancer Res. 55: 4293-4296.

Makino, Y., H. Tanaka, and I. Makino. 1994. Paradoxical derepression ofthe collagenase gene expression by the anti-rheumatic gold compoundaurothiomalate. Mol. Pharmacol. 46: 1084-1089.

Tanaka, H., Y. Makino, K.-D. Wright, J.-Å. Gustafsson, K. Okamoto, a ndI. Makino. 1995. Zinc ions antagonize the inhibitory effect ofaurothiomalate on glucocorticoid receptor function at physiologicalconcentrations. Mol. Pharmacol. 48: 938-945.

Sachi, Y., K. Hirota, H. Masutani, K. Toda, T. Okamoto, M. Takigawa, andJ. Yodoi. 1995. Induction of ADF/TRX by oxidative stress inkeratinocytes and lymphoid cells. Immunol. Lett. 44, 189-193.

Cappel, R. E., and H. F. Gilbert. 1988. Thiol/disulfide exchange between3-hydroxy-3-methyglutaryl-CoA reductase and glutathione. J. Biol. Chem.263: 12201-12212.

Snyder, G. H., M. J. Cennerazzo, A. J. Karalis, and D. Field. 1981.Electrostatic influence of local cysteine environments on disulfideexchange kinetics. Biochemistry 20: 6509-6518.

Xanthoudakis, S., G. Miao, F. Wang, Y.-C. E. Pan, and T. Curran. 1992.Redox-activation of Fos-Jun DNA binding activity is mediated by a DNArepair enzyme. EMBO J. 11: 3323-3335.

Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schütz, K.Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, and R. M. Evans.1995. The nuclear receptor superfamily: the second decade. Cell 83:835-839.

Qin, J., G. M. Clore, W. M. P. Kennedy, J. R. Huth, and A. M.Gronenborn. Solution structure of human thioredoxin in a mixed disulfideintermediate complex with its target peptide from the transcriptionfactor NFkB. Structure 3: 289-297.

Blake, M. J., R. Udelsman, G. J. Feulner, D. D. Norton, and N. J.Holbrook. 1991. Stress-induced heat shock protein 70 expression inadrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependentresponse. Proc. Natl. Acad. Sci. USA. 88: 9873-9877.

The role of NF-κB in HIV life cycle is critical especially in virusreactivation process within the latently infected cells has been widelyaccepted. After activation through intracellular signaling pathways suchas those elicited by T cell receptor antigen complex or by receptors forIL-1 or TNF, NF-κB initiates HIV gene expression by binding to thetarget DNA element within the promoter region of HIV LTR. Then, thevirus-encoded trans-activator Tat is produced and triggers explosiveviral replication. Since activation pathway of HIV gene expression bycellular transcription factor NF-κB conceptually precedes activation byviral trans-activators, it is conceptual to ascribe NF-κB as adeterminant of the maintenance and breakdown of the viral latency.Antioxidants may be effective in treating AIDS by blocking HIVreplication.

Another situation where NF-κB plays a role is hematogenic cancer cellmetastasis. NF-κB induces E-selectin (also known as ELAM-1) on thesurface of vascular endothelial cells. Since some cancer cellsconstitutively express a ligand for E-selectin, called sialyl-LewisXantigen, on their cell surface, induction of E-selectin is considered tobe a rate determining step of cancer cell-endothelial cell interaction.For example, when primary human umbilical venous endothelial cells(HUVEC) are treated with IL-1 or TNF, nuclear translocation of NF-κB isobserved, followed by the augmented expression of E-selectin. In onestudy, the cell-to-cell interaction between HUVEC and QG90 cell, a tumorcell line derived from human small cell carcinoma of the lung expressingsialyl-LewisX antigen was studied, and it was found that IL-1 was ableto induce the attachment of cancer cells to HUVEC. However, pretreatmentof HUVEC with N-acetylcysteine, aspirin or pentoxyphillin efficientlyblocked the cell-to-cell attachment in a dose-dependent manner. Okamoto,T. et al., Oxygen Radicals, Redox Regulation of the NF-kB Signaling andDisease Control by Antioxidants (poster), Proceedings of 3rd InternetWorld Congress on Biomedical Sciences, 1996.12.9-20 Riken, Tsukuba,Japan. See, also:

Ginn-Pease M E; Whisler R L. Redox signals and NF-kappaB activation in Tcells, Free Radic Biol Med. August 1998; 25 (3): 346-61.

Holmgren A. Thioredoxin. Ann Rev Biochem 1985; 54: 237-271.

Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem 1989; 264,13963-13966.

Ziegler D M. Role of reversible oxidation-reduction of enzymethios-disulfides in metabolic regulation. Ann Rev Biochem 1985; 54,305-329.

Allen J F. Redox control of transcription: sensors, response regulators,activators and repressors. FEBS Lett 1993; 332: 203-207.

Gilmore T D. NF-kappa B, KBF-1, dorsal and related matters. Cell 1990;62: 841-843.

Baeuerle P A. The inducible transcription activator NF-kappa B:regulation by distinct protein subunits. Biochim Biophys Acta 1991;1072: 63-80.

Baeuerle P A, Henkel T. Function and activation of NP-kappa B in theimmune system. Ann Rev mmunol 1994; 12: 141-179.

Thanos D, Maniatis T. NF-kappa B: a lesson in family values. Cell 1995;80: 529-532.

Schindler U, Baichwal V R. Three NF-kappa B binding sites in the humanE-selectin gene required for maxmal tumor necrosis factor alpha-inducedexpression. Mol Cell Biol 1994; 14: 5820-5831.

Okamoto T, Matsuyama T, Mori S, Hamamoto Y, Kobayashi N, Yamamoto N,Josephs F, Wong-Staal F, Shimotohno K. Augmentation of humanimmunodeficiency virus type 1 gene expression by tumor necrosis factoralpha. AIDS Res Hum Retrovir 1989, 5: 131-138.

Maekawa T, Itoh F, Okamoto T, Kurimoto M, Imamoto F, Shii S.Identification and purification of the enhancer-binding factor of humanimmunodeficiency virus-1. Multiple preteins and binding to otherenhancers. J Biol Chem 1989; 264: 2826-2831.

Stade B G, Messer G, Riethmuller G., Johnson J P. Structuralcharacteristics of the 5′ region of the human ICAM-1 gene. Immunobiol1990; 182: 79-87.

Mukaida N, Mahe Y, Matsushima K. Cooperative interaction of nuclearfactor-kappa B- and cis-regulatory enhancer binding protein-like factorbinding elements in activating the interleukin-8 gene bypro-inflammatory cytokines. J Biol Chem 1990; 265: 21128-21133.

Roebuck K A, Rahman A, Lakshminarayanan V, Janakidevi K, Malik A B. H2O2and tumor necrosis factor-alpha activate intercellular adhesion molecule1 (ICAM-1) gene transcription through distinct cis-regulatory elementswithin the ICAM-1 promoter. J Biol Chem 1995; 270: 18966-18974.

Donnelly R P, Crofford L J, Freeman S L, Buras J, Remmers E, Wilder R L,Fenton M J. Tissue-specific regulation of IL-6 production by IL-4.Differential effects of IL-4 on nuclear factor-kappa B activity inmonocytes and fibroblasts. J mmunol 1993; 151: 5603-5612.

Schreck R, Baeuerle P A. NF-kappa B as inducible transcriptionalactivator of the granulocyte-macrophage colony-stimulating factor gene.Mol Cell Biol 1990; 10: 12811286.

Staynov D Z, Cousins D J, Lee T H. A regulatory element in the promoterof the human granulocyte-macrophage colony-stimulating factor gene thathas related sequences in other T-cell-expressed cytokine genes. ProcNatl Acad Sci USA 1995; 92: 3606-3610.

Xie Q-W, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappaB/Rel in induction of nitric oxide synthase. J Biol Chem 1994; 269:4705-4708.

Sen R, Baltimore D. Inducibility of kappa immunoglobulinenhancer-binding protein NF-kappa B by a posttranslational mechanism.Cell 1986; 46: 705-716.

Nabel G, Baltimore D. An inducible transcription factor activatesexpression of human immunodeficiency virus in T cells. Nature 1987; 326:711-713.

Baeuerle P A, Baltimore D. Activation of DNA-binding activity in anapparently cytoplasmic precursor of the NF-kappa B transcription factor.Cell 1988A; 53: 211-217.

Baeuerle P A, Baltimore D. I-kappa B: a specific inhibitor of theNF-kappa B transcription factor. Science 1988B; 242: 540-546.

Ghosh S, Gifford A M, Riviere L R, Tempst P, Nolan G P, Baltimore D.Cloning of the p50 DNA binding subunit of NF-kappa B: homology to Reland dorsal. Cell 1990; 62: 1019-1029.

Ghosh S, Baltimore D. Activation in vitro of NF-kappa B byphosphorylation of its inhibitor I-kappa B. Nature 1990; 344: 678-682.

Read M A, Whitley M Z, Williams A J, Collins T. The proteasome pathwayis required for cytokine-induced endothelial-leukocyte adhesion moleculeexpression. J Exp Med 1994; 179: 503-512.

Hayashi T, Sekine T, Okamoto T. Identification of a new serine kinasethat activates NF-kappa B by direct phosphorylation. J Biol Chem 1993A:826: 26790-26795.

Shirakawa F, Mizel S B. In vitro activation and nuclear translocation ofNF-kappa B catalyzed by cyclic AMP-dependent protein kinase and proteinkinase C. Mol Cell Biol 1989; 9: 2424-2430.

Meichle A, Schutze S, Hensel G, Brunsing D, Kronke M. Protein kinaseC-independent activation of nuclear factor kB by tumor necrosis factor.J Biol Chem 1990; 265: 8339-8343.

Feuillard J, Gouy H, Bismuth G, Lee L M, Debre P, Korner M. Nf-kappa Bactivation by tumor necrosis factor alpha in the Jurkat T cell line isindependent of protein kinase A, protein kinase C, and Ca (2)-regulatedkinase. Cytokine 1991; 3: 257-265.

Ostrowski J, Sims J E, Sibley C H, Valentine M A, Dower S K, Meier K E,Bomsztyk K. A serine/threonine kinase activity is clsely associated witha 65-kDa phosphoprotein specifically recognized by the kappa B enhancerelement. J Biol Chem 1991; 266: 12722-12733.

Schutze S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, Kronke M.TNF activates NF-kappa B by phosphatidylcholine-specific phospholipaseC-induced “acidic” sphingomyelin breakdown. Cell 1992; 71: 765-776.

Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. Control ofI-kappa B-alpha proteolysis by site-specific, signal-inducedphosphorylation. Science 1995; 267: 1485-1488.

Cao Z, Henzel W J, Gao X. IRAK: a kinase associated with theinterleukin-1 receptor. Science 1996; 271: 1128-1131.

Chen Z J, Parent L, Maniatis T. Site-specific phosphorylation of IkBa bya novel ubiquitination-dependent protein kinase activity. Cell 1996; 84:853-862.

Naumann M, Scheidereit C. Activation of NF-kappa B in vivo is regulatedby mutiple phosphorylations. EMBO J 1994; 13: 4597-4607.

Li C-C H, Dai R-M, Chen E, Longo D L. Phosphorylation of NF-KB1-p50 isinvolved in NF-kappa B activation and stable DNA binding. J Biol Chem1994; 269: 30089-30092. 38. Okamoto T, Ogiwara H, Hayashi T, Mitsui A,Kawabe T, Yodoi J. Human thioredoxin/adult T cell leukemia-derivedfactor activates the enhancer binding protein of human immunodeficiencyvirus type 1 bt thiol redox control mechanism. Int Immunol 1992; 4:811-819.

Hayashi T, Ueno Y, Okamoto T. Oxidoreductive regulation of nuclearfactor kappa B. Involvement of a cellular reducing catalyst thioredoxin.J Biol Chem 1993B; 268: 11380-11388.

Tagaya Y, Maeda Y, Mitsui A, Kondo N, Matsui H, Hamuro J, Brown J, AraiK I, Yokota T, Wakasugi H, Yodoi J. ATL-derived factor (ADF), an IL-2receptor/Tac inducer homologous to thioredoxin: possible involvement ofdithiol-reduction in the IL-2 receptor induction. EMBO J 1989; 8:757-764.

Schreck R, Rieber P, Baeuerle P A. Reactive oxygen intermediates asapparently widely used messengers in the activation of the NF-kappa Btranscription factor and HIV-1. EMBO J 1991; 10: 2247-2258.

Molitor J A, Ballard D W, Greene W C. Kappa-B-specific DNA bindingproteins are differentially inhibited by enhancer mutations andbiological oxidation. New Biol 1991; 3: 987-996.

Toledano M B, Leonard W J. Modulation of transcription factor NF-kappa Bbinding activity by oxidation-reduction in vitro. Proc Natl Acad Sci USA1991; 88: 4328-4332. 44. Matthews J R, Wakasugi N, Virelizier J-L, YodoiJ, Hay R T. Thioredoxin regulates the DNA binding activity of NF-kappa Bby reduction of a disulfide bond involving cystein 62. Nucleic Acids Res1992; 20, 3821-3830.

Ghosh G, Van Duyne G, Ghosh S, Sigler P B. Structure of NF-kappa B p50homodimer bound to a kappa B site. Nature 1995; 373: 303-310.

Müller C W, Rey F A, Sodeoka M, Verdine G L, Harrison S C. Structure ofthe NF-kappa B p50 homodimer bound to DNA. Nature 1995; 373: 311-317.

Qin J, Clore G M, Kennedy W M P, Huth J R, Gronenborn A M. Solutionstructure of human thioredoxin in a mixed disulfide intermediate complexwith its target peptide from the transcription factor NF-kappa B.Structure 1995; 3: 289-297.

Roederer M, Staal F J T, Raju P A, Ela S W, Herzenberg L A, Herzenberg LA. Cytokine-stimulated human immunodeficiency virus replication isinhibited by N-acetyl-Lcysteine. Proc Natl Acad Sci USA 1990; 87:4884-4888.

Suzuki Y J, Aggarwal B B, Packer L. Alpha-lipoic acid is a potentinhibitor if NF-kappa B activation in human T cells. Biochem Biophys ResCommun 1992; 189: 1709-1715.

Meyer M, Schreck R, Baeuerle P A. H2O2 and antioxidants have oppositeeffects on activation of NF-kappa B and AP-1 in intact cells: AP-1 assecondary antioxidant-responsive factor. EMBO J 1993; 12: 2005-2015.

Biswas D K, Dezube B J, Ahlers C M, Pardee A B. Pentoxifylline inhibitsHIV-1 LTR-driven gene expression by blocking NF-kappa B action. J AIDS1993: 6: 778-786.

Suzuki Y J, Packer L. Signal transduction for nuclear factor-kappa Bactivation. Proposed location of antioxidant-inhibitable step. J Immunol1994; 153: 5008-5015.

Packer L, Witt E H, Tritschler H J. Alpha-lipoic acid as a biologicalantioxidant. Free Rad Biol Med 1995; 19: 227-250.

Sachi Y, Hirota K, Masutani H, Toda K, Okamoto T, Takigawa M, Yodoi J.Three NF-kappa B binding sites in the human E-selectin gene required formaximal tumor necrosis factor alpha-induced expression. Immunol Lett1995; 44: 189-193.

Yang J P, Merin J P, Nakano T, Kato T, Kitade Y, Okamoto T. Inhibitionof the DNA-binding activity of NF-kappa B by gold compounds in vitro.FEBS lett 1995; 361: 89-96.

Skosey J. L. in “Arthritis and allied conditions” (McCarty D J, KoopmanW J, eds) pp 603-614, Lea & Febiger, Philadelphia, 1993.

Insel P A. in “Autacoids: Drug Therapy of Inflammation” (Gilman G, etal, eds) pp. 670-681, Macmillan, New York, 1990.

Handel M L, McMorrow L B, Gravallese E. M. Nuclear factor-kB inrheumatoid synovium. Localization of p50 and p60. Arthritis Rheum 1996;38: 1762-1770.

Sakurada S, Kato T, Okamoto T. Induction of cytokines and ICAM-1 byproinflammatory cytokines in primary rheumatoid synovial fibroblasts andinhibition by N-acetyl-L-cysteine and aspirin. Int mmunol 1996 in press.

Bohnlein E, Lowenthal J W, Siekevitz M, Ballard D W, Franza B R, GreeneW C. The same inducible nuclear proteins regulates mitogen activation ofboth the interleukin-2 receptor-alpha gene and type 1 HIV. Cell 1988;53: 827-836.

Okamoto T, Benter T, Josephs S F, Sadaie M R, Wong-Staal F.Transcriptional activation from the long-terminal repeat of humanimmunodeficiency virus in vitro Virology 1990; 177: 606-614.

Arya S K, Guo C, Josephs S F, Wong-Staal F. Trans-activator gene ofhuman T-lymphotropic virus type III (HTLV-III). Science 1985; 229:69-73.

Sodroski J, Patarca R, Rosen C. Location of the trans-activating regionon the genome of human T-cell lymphotropic virus type III. Science 1985;229: 74-77.

Okamoto T, Wong-Staal F. Demonstration of virus-specific transcriptionalactivator(s) in cells infected with HTLV-III by an in vitro cell-freesystem. Cell 1986; 47: 29-35.

Peterlin B M, Luciw P A, Barr P J, Walker M D. Elevated levels of mRNAcan account for the trans-activation of human immunodeficiency virus.Proc Natl Acad Sci USA 1986; 83: 9734-9738.

Tozawa K, Sakurada S, Kohri K, Okamoto T. Effects of anti-nuclear factorkappa B reagents in blocking adhesion of human cancer cells to vascularendothelial cells. Cancer Res 1995; 55: 4162-4167.

Montgomery K F, Osborn L, Hession C, Tizard R, Goff D, Vassallo C, TarrP I, Bomsztyk K, Lobb R, Harlan J M, Pohlman T H. Activation ofendothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription.Proc Natl Acad Sci USA 1991; 88: 6523-6527.

Whelan J, Ghersa P, Huijsduijnen R H, Gray J, Chandra G, Talabot F,DeLamarter J F. An NF kappa B-like factor is essential but notsufficient for cytokine induction of endothelial leukocyte adhesionmolecule 1 (ELAM-1) gene transcription. Nuc Acid Res 1991; 19:2645-2653.

Dejana E, Bertocci F, Bortolami M C, Regonesi A, Tonta A, Breviario F,Giavazzi R. Interleukin 1 promotes tumor cell adhesion to cultured humanendothelial cells. J Clin nvest 1988; 82: 1466-1470.

Takada A, Ohmori K, Yoneda T, Tsuyuoka K, Hasegawa A, Kiso M, Kannagi R.Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis Xto adhesion of human cancer cells to vascular endothelium. Cancer Res1993; 53: 354-361.

Kira T, Merin J P, Baba M, Shigeta S, Okamoto T. Anti-Tat MTT assay: anovel anti-HIV drug screening system using the viral regulatory networkof replication. AIDS Res Hum Retrovir 1995; 11: 1359-1366.

Pigmented Epithelium Derived Factor

It is well known that solid tumors, such as carcinomas, requireneovascularization to continue growth beyond a few millimeters in size.This is because, as with all tissues, they need oxygen and must ridthemselves of toxic metabolic products. Further, rapidly growing tumorsmay have demands well in excess of that of normal tissues due to a highrate of cell replication. Therefore, one technique which has been soughtto be employed in fighting tumors is the use of pharmaceuticals andagents that block neovascularization, for example tumor necrosis factor,endostatin, angiostatin, and other agents. One agent that has arousedinterest is Pigmented Epithelium Derived Factor (PEDF), a protein of theserine protease inhibitor (serpin) supergene family, but withcharacteristics of a substrate rather than inhibitor. PEDF was named forits association with the pigmented RPE cells of the macula, describedabove. See:

Tombran-Tink et al., “Neuronal Differentiation of Retinoblastoma CellsInduced by Medium Conditioned by Human RPE Cells,” InvestigativeOphthalmology & Visual Science, 30 (8), 1700-1707 (1989);

G Chader, S P Becerra, L Johnson, J Tombran-Tink, F Steele and IRodriguez, PCT/US95/07201 filed Jun. 6, 1995, published under WO95/33480 on Dec. 14, 1995;

U.S. Ser. No. 07/952,796 entitled A DNA Clones for the Expression ofPigment Epithelium Derived Growth Factor and Related Proteins, filedSep. 24, 1992 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tinkand Sofia P. Becerra;

U.S. Ser. No. 08/257,963 entitled A Pigment Epithelium Derived Factor:Characterizations of Its Biological Activity and Sequences Encoding andExpressing the Protein, filed Jun. 7, 1994 by Gerald J. Chader, Sofia P.Becerra, Joan P. Schwartz, Takayuki Taniwaki and Yukihera Sugita, nowU.S. Pat. No. 5,840,686;

U.S. Ser. No. 08/279,979 entitled A Retinal Pigmented Epithelium DerivedNeurotrophic Factor, filed Jul. 25, 1994 by Fintan R. Steele, Gerald J.Chader, Joyce Tombran-Tink, Sofia P. Becerra and Ignacio R. Rodriquezand Lincoln Johnson;

U.S. Ser. No. 08/367,841 entitled A Pigment Epithelium Derived Factor:Characterization, Genomic Organization and Sequence of the PEDF Gene,filed Dec. 30, 1994 by Gerald J. Chader, Joyce Tombran-Tink, Sofia P.Becerra, Ignacio R. Rodriquez and Fintan R. Steele and Lincoln Johnson;

U.S. Ser. No. 08/377,710 entitled A DNA Clones for the Expression ofPigment Epithelium Derived Factor and Related Proteins, filed Jan. 25,1995 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink, Sofia P.Becerra and Ignacio R. Rodriquez;

U.S. Ser. No. 08/520,373 entitled A Retinal Pigmented Epithelium DerivedNeurotrophic Factor, filed Aug. 29, 1995 by Gerald J. Chader, JoyceTombran-Tink, Sofia P. Becerra, Ignacio R. Rodriquez and Fintan R.Steele;

F R Steele, G J Chader, L V Johnson, J Tombran-Tink. Pigmentepithelium-derived factor: neurotrophic activity and identification as amember of the serine protease inhibitor gene family. Proc. Natl. Acad.Sci. U.S.A. 1993, 90, 1526-1530;

S P Becerra, I Palmer, A Kumar, F Steele, J Shiloach, V Notario, G JChader. Overexpression of fetal human pigment epithelium-derived factorin Escherichia coil: a functionally active neurotrophic factor. J BiolChem Nov. 5, 1993; 268 (31): 23148-56;

Perez-Mediavilla L A, Chew C, Campochiaro P A, Nickells R W, Notario V,Zack D J, Becerra S P. Sequence and expression analysis of bovinepigment epithelium-derived factor. Biochim Biophys Acta Jun. 16, 1998;1398 (2): 203-14;

Slavc I; Rodriguez I R; Mazuruk K; Chader G J; Biegel J A. Mutationanalysis and loss of heterozygosity of PEDF in central nervous systemprimitive neuroectodermal tumors, Int J Cancer 1997; 72 (2): 277-82.

PEDF is a potent autocrine and paracrine hormone which blocks epithelialcell proliferation (including vascular epithelial cells, necessary forneovascularization), and promotes cellular differentiation, and isneurotrophic and neuroprotective. Sugita Y, Becerra S P, Chader G J,Schwartz J P, Pigment epithelium-derived factor (PEDF) has directeffects on the metabolism and proliferation of microglia and indirecteffects on astrocytes., J Neurosci Res Sep. 15, 1997; 49 (6): 710-8.Subsequent studies have confirmed that PEDF or its isoforms are widelydistributed throughout the body, but with relatively high concentrationin the pigmented epithelial cells of the retina and central nervoussystem. PEDF may help cells resist apoptosis. Araki T, Taniwaki T,Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor(PEDF) differentially protects immature but not mature cerebellargranule cells against apoptotic cell death, J Neurosci Res Jul. 1, 1998;53 (1): 7-15. Glutathione depletion has also been directly associatedwith failure of differentiation. Esposito F, Agosti V, Morrone G, MorraF, Cuomo C, Russo T, Venuta S, Cimino F, Inhibition of thedifferentiation of human myeloid cell lines by redox changes inducedthrough glutathione depletion, Biochem. J. (1994) 301, 649-653.

PEDF binds to extracellular matrixes. Alberdi E, Hyde C C, Becerra S P,Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans:analysis of the binding site. Biochemistry Jul. 28, 1998; 37 (30):10643-52 (Published erratum appears in Biochemistry Dec. 22, 1998; 37(51): 18128).

PEDF is among the most potent direct angiogenesis factors known. In theeye, it prevents ingrowth of blood vessels in the lens, retina andvitreous body of the eye. Ortego J, Escribano J, Becerra S P,Coca-Prados M, Gene expression of the neurotrophic pigmentepithelium-derived factor in the human ciliary epithelium. Synthesis andsecretion into the aqueous humor, Invest Ophthalmol Vis Sci December1996; 37 (13): 2759-67. PEDF is a dramatic enhancer of cellulardifferentiation, and is capable, for example, of inducing retinoblastomacells to retrotransform into normal appearing cells. Stratikos E,Alberdi E, Gettins P G, Becerra S P, Recombinant human pigmentepithelium-derived factor (PEDF): characterization of PEDF overexpressedand secreted by eukaryotic cells. Protein Sci December 1996; 5 (12):2575-82. PEDF protects neural tissue against an array of injuriousfactors, for example, against the excitatory neurotoxicity of glutamate.Taniwaki T, Hirashima N, Becerra S P, Chader G J, Etcheberrigaray R,Schwartz J P. Pigment epithelium-derived factor protects culturedcerebellar granule cells against glutamate-induced neurotoxicity. JNeurochem January 1997; 68 (1): 26-32.

PEDF is produced by the stromal cells of the endometrium and has astrong effect on the growth and differentiation of the glandularepithelium. When stromal cells become deciduous cells, in response tohormones and pregnancy, PEDF production is considered crucial to prevent(i) uncontrolled growth and penetration of the otherwise highly invasivetrophoblastic cells of the placenta, into the uterine wall, and (ii)uncontrolled ingrowth of the blood vessels from the chorionic villi,into the uterine wall.

PEDF controls the cell cycle in many different cell types, by a directeffect on cell cycle control factors. The source of PEDF, namely theretinal pigment epithelium (RPE), may be crucial to the normaldevelopment and function of the neural retina. A variety of biologicallyactive molecules, including growth factors, are synthesized and secretedby RPE cells. The RPE develops prior to and lies adjacent to the neuralretina, and that it functions as part of the blood-retina barrier. Fineet al., The Retina, Ocular Histology: A Text and Atlas, New York, Harper& Row, 61-70 (1979), the RPE has been implicated in vascular,inflammatory, degenerative, and dystrophic diseases of the eye. Elner etal., Am. J. Pathol., 136, 745-750 (1990). In addition to growth factors,nutrients and metabolites are also exchanged between the RPE and theretina. For example, the RPE supplies to the retina the well-knowngrowth factors PDGF, FGF, TGFα, and TGFβ. Campochiaro et al., Invest.Ophthalmol. Vis. Sci., 29, 305-311 (1988): Plouet, Invest. Ophthalmol.Vis. Sci., 29, 106-114 (1988); Fassio et al., Invest. Ophthalmol. Vis.Sci., 29, 242-250 (1988); Connor et al., Invest. Ophthalmol. Vis. Sci.,29, 307-313 (1988). It is very likely that these and other unknownfactors supplied by the RPE influence the organization, differentiation,and normal functioning of the retina.

In order to study and determine the effects of putative differentiationfactors secreted by the RPE, cultured cells have been subjected toretinal extracts and conditioned medium obtained from cultures of humanfetal RPE cells. For example, U.S. Pat. No. 4,996,159 (Glaser) disclosesa neovascularization inhibitor recovered from RPE cells that is of amolecular weight of about 57,000±3,000. Similarly, U.S. Pat. Nos.1,700,691 (Stuart), 4,477,435 (Courtois et al.), and 4,670,257 (Guedonborn Saglier et al.) disclose retinal extracts and the use of theseextracts for cellular regeneration and treatment of ocular disease.Furthermore, U.S. Pat. Nos. 4,770,877 (Jacobson) and 4,534,967 (Jacobsonet al.) describe cell proliferation inhibitors purified from theposterior portion of bovine vitreous humor.

PEDF has been isolated from human RPE as a 50-kDa protein. Tombran-Tinket al., Invest. Ophthalmol. Vis. Sci., 29, 414 (1989); Tombran-Tink etal., Invest. Ohthalmol. Vis. Sci., 30, 1700-1707 (1989); Tombran-Tink etal., “PEDF: A Pigment Epithelium-derived Factor with Potent NeuronalDifferentiative Activity,” Experimental Eye Research, 53, 411-414(1991). Specifically, PEDF has been demonstrated to induce thedifferentiation of human Y79 retinoblastoma cells, which are aneoplastic counterpart of normal retinoblasts. Chader, Cell Different.,20, 209-216 (1987); Taniwaki T, Becerra S P, Chader G J, Schwartz J P,Pigment epithelium-derived factor is a survival factor for cerebellargranule cells in culture, J Neurochem June 1995; 64 (6): 2509-17. Thedifferentiative changes induced by PEDF include the extension of acomplex meshwork of neurites, and expression of neuronal markers such asneuron-specific enolase and neurofilament proteins. This is why thesynthesis and secretion of PEDF protein by the RPE is believed toinfluence the development and differentiation of the neural retina.Furthermore, PEDF is only highly expressed in undifferentiated humanretinal cells, like Y79 retinoblastoma cells, but is either absent ordownregulated in their differentiated counterparts. It was also reportedthat PEDF mRNA is expressed in abundance in quiescent human fetal W1fibroblast cells and not expressed in their senescent counterparts.Pignolo et al. (1993), J. Biol. Chem., 268: 2949-295.

Further study of PEDF and examination of its potential therapeutic usein the treatment of inflammatory, vascular, degenerative, and dystrophicdiseases of the retina and central nervous system (CNS) necessitates theobtention of large quantities of PEDF. Unfortunately, the low abundanceof PEDF in fetal human eye and, furthermore, the rare availability ofits source tissue, especially in light of restrictions on the use offetal tissue in research and therapeutic applications, make furtherstudy of PEDF difficult at best. Therefore, a recombinant technique wasdeveloped to procure a supply of the factor. See, U.S. Pat. No.5,840,686, supra.

Based upon the protein amino acid sequence, PEDF has been found to haveextensive sequence homology with the serpin gene family, members ofwhich are serine protease inhibitors. Many members of this family have astrictly conserved domain at the carboxyl terminus which serves as thereactive site of the protein. These proteins are thus thought to bederived from a common ancestral gene. However the developmentalregulation differs greatly among members of the serpin gene family andmany have deviated from the classical protease inhibitory activity.Becerra S P, Structure-function studies on PEDF. A noninhibitory serpinwith neurotrophiic activity, Adv Exp Med Biol 1997; 425: 223-37.Although PEDF shares sequence homology with serpins, analysis of thecDNA sequence indicates that it lacks the conserved domain and thus maynot function as a classical prolease inhibitor.

Genomic sequencing and analysis of PEDF has provided sequences ofintrons and exons as well as approx. 4 kb of 5′-upstream sequence. Thegene for PEDF has been localized to 17p13.1 using both in situhybridization and analyses of somatic cell hybrid panels. Tombran-Tink,et al., (1994) Genomics, 19: 266-272. This is very close to the p53tumor suppressor gene as well as to the chromosomal localization of anumber of hereditary cancers unrelated to mutations in the p53 geneproduct PEDF thus becomes a prime candidate gene for these cancers.

Although PEDF is particularly highly expressed by RPE cells, it isdetectable in most tissues, cell types, tumors, etc. by Northern andWestern blot analyses. It is readily detected, for example in vitreousand aqueous humors. The important question of subcellular localizationof PEDF has also been addressed. Although the bulk of the PEDF appearsto be secreted, PEDF is also associated with the nucleus as well as withvery specific cytoskeletal structures in the cytoplasm. Importantly,this varies as to the age of the cells and the specific cell-cyclestate. For example, the protein appears to concentrate at the tips ofthe pseudopods of primate RPE cells that interact with the substratumduring the initial stages of attachment. Later though, this stainingdisappears and there is appearance of the protein in association withspecific cytoskeletal structures and the nucleus. Thus it appears thatPEDF plays an important intracellular role in both nucleus andcytoplasm.

There is PEDF expression in dividing, undifferentiated Y-79 cells andlittle or no expression in their quiescent, differentiated counterparts.Tombran-Tink, et al., (1994) Genomics, 19: 266-272. The synthesis ofPEDF in WI-38 fibroblast cells is restricted to the G₀ stage of the cellcycle in young cells. Pignolo et al. (1993), J. Biol. Chem., 268:2949-295. Moreover, in old senescent cells, PEDF messenger RNA isabsent.

In the retina, PEDF inhibits the Muller glial cells. Since Muller cellsare similar to astroglia, PEDF would be similarly effective in blockinggliosis in conditions such as retinal etachment, diabetes, RetinitisPigmentosa, etc. as well as sparing the lives of the retinal neurons.Thus, administration of glutathione, to alter cellular redox potential,and thereby alter PEDF expression, may have particular value.

Apparently, in macular degeneration, the pigmented RPE cells becomedefective, and die, resulting in a functional loss of PEDF in themacula. Without the continuous presence of PEDF, vascular epithelialcells undergo a de-differentiation and enter into a proliferative stage,resulting in neovascularization, with invasion of the cornea in vitreouswith blood vessels. The amount of inhibitory PEDF produced by retinalcells is positively correlated with oxygen concentration. Thus, PEDF ispresumed to play a role in ischemia-driven retinal neovascularization.In fact, studies have shown that it is not necessary to kill the RPEcells to reduce PEDF availability. The availability of PEDF is sensitiveto the redox potential of the cell, being more available in a reducedstate and less available when the cell is in an oxidized state.(Ischemia is associated with a state in which cells produce an excess offree radicals. These may be due to exhaustion of antioxidants, celldeath or apoptosis, or accumulation of toxic metabolic waste). Thisfeedback regulation, which is applicable to other PEDF producing cells,thus induces vascularization where blood flow is needed (relativelyoxidized redox potential) while maintaining an appropriate balance andallowing certain privileged tissues to remain unvascularized or withhighly controlled vascularization. The oxidative control over PEDF isbelieved to be at the translative or post-translative levels, as mRNAlevels are generally unchanged. It is noted that other classes ofbiologically active agents respond to redox state throughtranscriptional modification or sensitivity.

Efforts to directly administer PEDF, a peptide hormone, are met withdifficulty, due to both the unavailability of bulk quantities of PEDFand difficulties in administration thereof.

Alberdi E, et al., Binding of Pigment Epithelium-derived Factor (PEDF)to Retinoblastoma Cells and Cerebellar Granule Neurons, Evidence for apedf receptor. J Biol Chem. Oct. 29, 1999; 274 (44): 31605-31612.

Cao W, et al., Pigment epithelium-derived factor protects culturedretinal neurons against hydrogen peroxide-induced cell death. J.Neurosci Res. Sep. 15, 1999; 57 (6): 789-800.

Houenou L J, et al., Pigment epithelium-derived factor promotes thesurvival and differentiation of developing spinal motor neurons. J CompNeurol. Sep. 27, 1999; 412 (3): 506-14.

Bilak M M, et al., Pigment epithelium-derived factor (PEDF) protectsmotor neurons from chronic glutamate-mediated neurodegeneration. JNeuropathol Exp Neurol. July 1999; 58 (7): 719-28.

Koenekoop R. et al., Four polymorphic variations in the PEDF geneidentified during the mutation screening of patients with Lebercongenital amaurosis., Mol Vis. Jul. 2, 1999; 5: 10.

Dawson D W, et al., Pigment epithelium-derived factor: a potentinhibitor of angiogenesis. Science. Jul. 9, 1999; 285 (5425): 245-8.

DeCoster M A, et al., Neuroprotection by pigment epithelial-derivedfactor against glutamate toxicity in developing primary hippocampalneurons. J Neurosci Res. Jun. 15, 1999; 56 (6): 604-10.

Tresini M, et al., Effects of donor age on the expression of a marker ofreplicative senescence (EPC-1) in human dermal fibroblasts. J CellPhysiol. April 1999, 179 (1): 11-7.

Palmieri D, et al., Age-related expression of PEDF/EPC-1 in humanendometrial stromal fibroblasts: implications for interactivesenescence. Exp Cell Res. Feb. 25, 1999; 247 (1): 142-7.

Malchiodi-Albedi F, et al., PEDF (pigment epithelium-derived factor)promotes increase and maturation of pigment granules in pigmentepithelial cells in neonatal albino rat retinal cultures. Int J DevNeurosci. August 1998; 16 (5): 423-32.

Alberdi E, et al., Pigment epithelium-derived factor (PEDF) binds toglycosaminoglycans: analysis of the binding site. Biochemistry. Jul. 28,1998: 37 (30): 10643-52.

Perez-Mediavilla L A, et al., Sequence and expression analysis of bovinepigment epithelium-derived factor. Biochim Biophys Acta. Jun. 16, 1998;1398 (2): 203-14.

Araki T, et al., Pigment epithelium-derived factor (PEDF) differentiallyprotects immature but not mature cerebellar granule cells againstapoptotic cell death. J Neurosci Res. Jul. 1, 1998; 53 (1): 7-15.

Carwile M E, et al., Rod outer segment maintenance is enhanced in thepresence of bFGF, CNTF and GDNF. Exp Eye Res. June 1998; 66 (6):791-805.

Kozaki K, et al., Isolation, purification, and characterization of acollagen-associated serpin, caspin, produced by murine colonadenocarcinoma cells. J Biol Chem. Jun. 12, 1998; 273 (24): 15125-30.

Singh V K, et al., Structural and comparative analysis of the mouse genefor pigment epithelium-derived factor (PEDF). Mol Vis. Apr. 20, 1998; 4:7.

Broekhuyse R M, et al., Differential effect of macrophage depletion ontwo forms of experimental uveitis evoked by pigment epithelial membraneprotein (EAPU), and by melanin-protein (EMIU). Exp Eye Res. December1997; 65 (6): 841-8.

Becerra S P, Structure-function studies on PEDF. A noninhibitory serpinwith neurotrophic activity. Adv Exp Med Biol. 1997; 425: 223-37. Review.

Sugita Y, et al., Pigment epithelium-derived factor (PEDF) has directeffects on the metabolism and proliferation of microglia and indirecteffects on astrocytes. J Neurosci Res. Sep. 15, 1997; 49 (6): 710-8.

Slavc I, et al., Mutation analysis and loss of heterozygosity of PEDF incentral nervous system primitive neuroectodermal tumors. Int J Cancer.Jul. 17, 1997; 72 (2): 277-82.

Taniwaki T, et al., Pigment epithelium-derived factor protects culturedcerebellar granule cells against glutamate-induced neurotoxicity. JNeurochem. January 1997; 68 (1): 26-32.

Ortego J, et al., Gene expression of the neurotrophic pigmentepithelium-derived factor in the human ciliary epithelium. Synthesis andsecretion into the aqueous humor. Invest Ophthalmol Vis Sci. December1996; 37 (13): 2759-67.

Stratikos E, et al., Recombinant human pigment epithelium-derived factor(PEDF): characterization of PEDF overexpressed and secreted byeukaryotic cells. Protein Sci. December 1996; 5 (12): 2575-82.

Tombran-Tink J, et al., Organization, evolutionary conservation,expression and unusual Alu density of the human gene for pigmentepithelium-derived factor, a unique neurotrophic serpin. Mol Vis. Nov.4, 1996; 2: 11.

Wu Y Q, et al., Proteolytic activity directed toward pigmentepithelium-derived factor in vitreous of bovine eyes. Implications ofproteolytic processing. Invest Ophthalmol Vis Sci. September 1996; 37(10): 1984-93.

Goliath R, et al., The gene for PEDF, a retinal growth factor is a primecandidate for retinitis pigmentosa and is tightly linked to the RP13locus on chromosome 17p13.3. Mol Vis. Jun. 19, 1996; 2: 5.

Lotery A J, et al., Localisation of a gene for central areolar choroidaldystrophy to chromosome 17p. Hum Mol Genet. May 1996; 5 (5): 705-8.

Phillips N J, et al., Allelic deletion on chromosome 17p13.3 in earlyovarian cancer. Cancer Res. Feb. 1, 1996; 56 (3): 606-11.

Balciuniene J, et al., A gene for autosomal dominant progressive conedystrophy (CORD5) maps to chromosome 17p12-p13. Genomics. Nov. 20, 1995;30 (2): 281-6.

Becerra S P, et al., Pigment epithelium-derived factor behaves like anoninhibitory serpin. Neurotrophic activity does not require the serpinreactive loop. J Biol Chem. Oct. 27, 1995; 270 (43): 25992-9.

DiPaolo B R, et al., Identification of proteins differentially expressedin quiescent and proliferatively senescent fibroblast cultures. Exp CellRes. September 1995; 220 (1): 178-85.

Wu Y Q, et al., Identification of pigment epithelium-derived factor inthe interphotoreceptor matrix of bovine eyes. Protein Expr Purif. August1995; 6 (4): 447-56.

Tombran-Tink J. et al., Expression, secretion, and age-relateddownregulation of pigment epithelium-derived factor, a serpin withneurotrophic activity. J Neurosci. July 1995; 15 (7 Pt 1): 4992-5003.

Taniwaki T, et al., Pigment epithelium-derived factor is a survivalfactor for cerebellar granule cells in culture. J Neurochem. June 1995;64 (6): 2509-17.

Pignolo R J, et al., Analysis of EPC-1 growth state-dependentexpression, specificity, and conservation of related sequences. J CellPhysiol. January 1995; 162 (1): 110-8.

Tombran-Tink J, et al., Localization of the gene for pigmentepithelium-derived factor (PEDF) to chromosome 17p13.1 and expression incultured human retinoblastoma cells. Genomics. Jan. 15, 1994; 19 (2):266-72.

Seigel G M, et al., Differentiation of Y79 retinoblastoma cells withpigment epithelial-derived factor and interphotoreceptor matrix wash:effects on tumorigenicity. Growth Factors. 1994; 10 (4): 289-97.

Becerra S P, et al., Overexpression of fetal human pigmentepithelium-derived factor in Escherichia coli. A functionally activeneurotrophic factor. J Biol Chem. Nov. 5, 1993; 268 (31): 23148-56.

Pignolo R J, et al., Senescent WI-38 cells fail to express EPC-1, a geneinduced in young cells upon entry into the G0 state. J Biol Chem. Apr.25, 1993; 268 (12): 8949-57.

Steele F R, et al., Pigment epithelium-derived factor: neurotrophicactivity and identification as a member of the serine protease inhibitorgene family. Proc Natl Acad Sci USA. Feb. 15, 1993; 90 (4): 1526-30.

Tombran-Tink J, et al., PEDF: a pigment epithelium-derived factor withpotent neuronal differentiative activity. Exp Eye Res. September 1991;53 (3): 411-4.

Seigel et al., Growth Factors, vol. 10, pp. 289-297, 1994.

Becerra et al., “Recombinant Human Fetal Retinal PigmentEpithelium-Derived Factor (PEDF).” Abstract 658-50, presented atInvestigative Ophthalmology & Visual Science Annual Meeting (May 3-May8, 1992).

Becerra et al., “A Novel Retinal Neurotrophic Factor (PEDF): A SerineProtease Inhibitor?” presented at NIH Research Festival 1992 (Sep.21-25, 1992).

Becerra, et al., “Structure-Function Studies of Pigment EpitheliumDerived Factor (PEDF),” The FASEB Journal (Abstract No. 192), vol. 7,No. 7, Apr. 20, 1993.

Pignolo, R. J., et al., “Senescent WI-38 Cells Fail To Express EPC-1, AGene Induced In Young Cells Upon Entry Into The G.sub.0 State,” TheJournal of Biological Chemistry, vol. 268, No. 12, Apr. 25, 1993, pp.8949-8957.

Tombran-Tink et al., “RPE-54—A Unique RPE Product with NeuronalDifferentiating Activity,” Investigative Ophthalmology & Visual Science,29, 414 (1989).

Tombran-Tink et al., “Molecular Cloning and Chromoscomal Localization ofthe Gene for Human Pigment Epithelium-Derived Factor (PEDF),”Investigative Ophthalmology & Visual Science, 33 (4), 828 (1992).

Tombran-Tink, et al., “Neurotrophic Activity of InterphotoreceptorMatrix on Human Y79 Retinoblastoma Cells,” The Journal of ComparativeNeurology, 1992.

Zhiqiang Zou, et al., “Maspin, A Serpin With Tumor-Suppressing ActivityIn Human Mammary Epithelial Cells,” Science, vol. 263, pp. 526-530, Jan.28, 1994.

Metabolism of Glutathione.

The synthesis of GSH is dependent upon the availability of cysteineeither supplied directly from the diet or cysteine or indirectly frommethionine via the transsulfuration pathway. GSH synthesis andmetabolism is governed by the enzymes of the γ-glutamyl cycle. GSH issynthesized intracellularly by the consecutive actions ofγ-glutamylcysteinyl synthetase (Reaction 1) and GSH synthetase (Reaction2). The action of the latter enzyme is feedback inhibited by GSH. Thebreakdown of GSH (and also of its oxidized form, GSSG) is catalyzed byγ-glutamyl transpeptidase, which catalyzes the transfer of thegamma-glutamyl moiety to acceptors such as sulfhydryl-containing aminoacids, certain dipeptides, and GSH itself (Reaction 3). The cellularturnover of GSH is associated with its transport, in the form of GSH,across cell membranes, where the majority of the transpeptidase isfound. During this transport, GSH interacts with γ-glutamyl transferase(also known as transpeptidase) to form γ-glutamyl amino acids which aretransported into cells. Intracellular γ-glutamyl amino acids aresubstrates of γ-glutamyl cyclotransferase (Reaction 4) which convertsthese compounds into the corresponding amino acids and 5-oxo-L-proline.The ATP-dependent conversion of 5-L-oxoproline to L-glutamate iscatalyzed by the intracellular enzyme 5-oxo-prolinase (Reaction 5). Thecysteinylglycine formed in the transpeptidase reaction is split bydipeptidase (Reaction 6). These six reactions constitute the γ-glutamylcycle, which accounts for the synthesis and enzymatic degradation ofGSH.

Two of the enzymes of the cycle also function in the metabolism ofS-substituted GSH derivatives, which may be formed nonenzymatically byreaction of GSH with certain electrophilic compounds or by GSHS-transferases (Reaction 7). The γ-glutamyl moiety of such conjugates isremoved by the action of γ-glutamyl transpeptidase (Reaction 3), areaction facilitated by γ-glutamyl amino acid formation. The resultingS-substituted cysteinylglycines are cleaved by dipeptidase (Reaction 6A)to yield the corresponding S-substituted cysteines, which may undergoN-acetylation (Reaction 8) or an additional transpeptidation reaction toform the corresponding γ-glutamyl derivative (Reaction 3A).

Intracellular GSH is converted to its oxidized, dimeric form (GSSG) byselenium-containing GSH peroxidase, which catalyzes the reduction ofH₂O₂ and other peroxides (Reaction 9). GSH is also converted to GSSG bytranshydrogenation (Reaction 10). Reduction of GSSG to GSH is mediatedby the widely-distributed enzyme GSSG reductase which uses NADPH(Reaction 11). Extracellular conversion of GSH to GSSG has also beenreported; the overall reaction requires O₂ and leads to the formation ofH₂O₂ (Reaction 12). GSSG is also formed by reaction of GSH with freeradicals. The glutathione-dependent antioxidant system consists ofglutathione plus two enzymes: glutathione peroxidase and glutathionereductase. As this system operates, glutathione cycles between itsoxidized (GSSG) and reduced (GSH) forms.

Lipid hydroperoxides, which are formed during the peroxidation of lipidscontaining unsaturated fatty acids, are reduced, not by the usualglutathione peroxidase, but by a special enzyme designed specifically tohandle peroxidized fatty acids in phospholipids. This enzyme, known asphospholipid hydroperoxide glutathione peroxidase is protein that canreduce both H₂O₂ and lipid hydroperoxides to the correspondinghydroxides (water and a lipid hydroxide, respectively). In contrast tothe phospholipid hydroperoxide glutathione peroxidase, ordinaryglutathione peroxidase is unable to act on lipid hydroperoxides.

Transport of Glutathione.

The intracellular level of GSH in mammalian cells is in the range of0.5-10 millimolar, while micromolar concentrations are typically foundin blood plasma. Intracellular glutathione is normally over 99% reducedform (GSH). The normal healthy adult human liver synthesizes 8-10 gramsof GSH daily. Normally, there is an appreciable flow of GSH from liverinto plasma. The major organs involved in the inter-organ transport ofGSH are the liver and the kidney, which is the primary organ forclearance of circulating GSH. It has been estimated to account for50-67% of net plasma GSH turnover. Several investigators have found thatduring a single pass through the kidney, 80% or more of the plasma GSHis extracted, greatly exceeding the amount which could be accounted forby glomerular filtration. While the filtered GSH is degraded stepwise bythe action of the brush-border enzymes γ-glutamyltransferase andcysteinylglycine dipeptidase, the remainder of the GSH appears to betransported via an unrelated, Na+-dependent system present inbasal-lateral membranes.

GSH transported from hepatocytes interacts with the transpeptidase ofductile cells, and there appears to be a substantial reabsorption ofmetabolites by ductule endothelium. In the rat, about 12 and 4nmoles/g/min of GSH appear in the hepatic vein and bile, respectively.

Glutathione exists in plasma in four forms: reduced glutathione (GSH),oxidized glutathione (GSSG), mixed disulfide with cysteine (CySSG) andprotein bound through a sulfhydryl linkage (GSSPr). The distribution ofglutathione equivalents is significantly different than that ofcyst(e)ine, and when either GSH or cysteine is added at physiologicalconcentration, a rapid redistribution occurs. The distribution ofglutathione equivalents in rat plasma is 70.0% protein bound, with theremaining 30% apportioned as follows: 28.0% GSH, 9.5% GSSG, and 62.6% asthe mixed disulfide with cysteine. The distribution of cysteineequivalents was found to be 23% protein bound, with the remaining 77%distributed as follows: 5.9% cysteine, 83.1% cystine, and 10.8% as themixed disulfide with glutathione. Plasma thiols and disulfides are notin equilibrium, but appear to be in a steady state maintained in part bytransport of these compounds between tissues during the interorgan phaseof their metabolism. The large amounts of protein-bound glutathione andcysteine provide substantial buffering which must be considered in theanalysis of transient changes in glutathione and cysteine. Thisbuffering may protect against transient thiol-disulfide redox changeswhich could affect the structure and activity of plasma and plasmamembrane proteins. In erythrocytes, GSH has been implicated in reactionswhich maintain the native structure of hemoglobin and of enzymes andmembrane proteins. GSH is present in erythrocytes at levels 1000 timesgreater than in plasma. It functions as the major small moleculeantioxidant defense against toxic free radicals, an inevitableby-product of the erythrocytes' handling of O₂.

Glutathione and the Immune System.

The importance of thiols and especially of GSH to lymphocyte functionhas been known for many years. Adequate concentrations of GSH arerequired for mixed lymphocyte reactions, T-cell proliferation, T- and B-cell differentiation, cytotoxic T-cell activity, and natural killer cellactivity. Adequate GSH levels have been shown to be necessary formicrotubule polymerization in neutrophils. Intraperitoneallyadministered GSH augments the activation of cytotoxic T-lymphocytes inmice, and dietary GSH was found to improve the splenic status of GSH inaging mice, and to enhance T-cell-mediated immune responses.

The presence of macrophages can cause a substantial increase of theintracellular GSH levels of activated lymphocytes in their vicinity.Macrophages consume cystine via a strong membrane transport system, andgenerate large amounts of cysteine which they release into theextracellular space. It has been demonstrated that macrophage GSH levels(and therefore cysteine equivalents) can be augmented by exogenous GSH.T-cells cannot produce their own cysteine, and it is required by T-cellsas the rate-limiting precursor of GSH synthesis. The intracellular GSHlevel and the DNA synthesis activity in mitogenically-stimulatedlymphocytes are strongly increased by exogenous cysteine, but notcystine. In T-cells, the membrane transport activity for cystine isten-fold lower than that for cysteine. As a consequence, T-cells have alow baseline supply of cysteine, even under healthy physiologicalconditions. The cysteine supply function of the macrophages is animportant part of the mechanism which enables T-cells to shift from aGSH-poor to a GSH-rich state.

The importance of the intracellular GSH concentration for the activationof T-cells is well established. It has been reported that GSH levels inT-cells rise after treatment with GSH; it is unclear whether thisincrease is due to uptake of the intact GSH or via extracellularbreakdown, transport of breakdown products, and subsequent intracellularGSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cellactivation in vitro. Depletion of intracellular GSH has been shown toinhibit the mitogenically-induced nuclear size transformation in theearly phase of the response. Cysteine and GSH depletion also affects thefunction of activated T-cells, such as cycling T-cell clones andactivated cytotoxic T-lymphocyte precursor cells in the late phase ofthe allogenic mixed lymphocyte culture. DNA synthesis and proteinsynthesis in IL-2 dependent T-cell clones, as well as the continuedgrowth of preactivated CTL precursor cells and/or their functionaldifferentiation into cytotoxic effector cells are strongly sensitive toGSH depletion.

The activation of physiologic activity of mouse cytotoxic T-lymphocytesin vivo was found to be augmented by interperitoneal (i.p.) GSH in thelate phase but not in the early phase of the response. The injection ofGSH on the third day post immunization mediated a 5-fold augmentation ofcytotoxic activity. Dietary GSH supplementation can reverseage-associated decline of immune response in rats, as demonstrated bymaintenance of Concanavalin A stimulated proliferation of splenocytes inolder rats.

Glutathione status is a major determinant of protection againstoxidative injury. GSH acts on the one hand by reducing hydrogen peroxideand organic hydroperoxides in reactions catalyzed by glutathioneperoxidases, and on the other hand by conjugating with electropililicxenobiotic intermediates capable of inducing oxidant stress. Theepithelial cells of the renal tubule have a high concentration of GSH,no doubt because the kidneys function in toxin and waste elimination,and the epithelium of the renal tubule is exposed to a variety of toxiccompounds. GSH, transported into cells from the extracellular medium,substantially protects isolated cells from intestine and lung areagainst t-butylhydroperoxide, menadione or paraquat-induced toxicity.Isolated kidney cells also transport GSH, which can supplementendogenous synthesis of GSH to protect against oxidant injury. HepaticGSH content has also been reported to rise, indeed to double, in thepresence of exogenous GSH. This may be due either to direct transport,as has been reported for intestinal and alveolar cells, or viaextracellular degradation, transport, and intracellular resynthesis.

The nucleophilic sulfur atom of the cysteine moiety of GSH serves as amechanism to protect cells from harmful effects induced by toxicelectrophiles. The concept that glutathione S-conjugate biosynthesis isan important mechanism of drug and chemical detoxification is wellestablished. GSH conjugation of a substrate generally requires both GSHand glutathione-S-transferase activity. The existence of multipleglutathione-S-transferases with specific, but also overlapping,substrate specificities enables the enzyme system to handle a wide rangeof compounds.

Several classes of compounds are believed to be converted by glutathioneconjugate formation to toxic metabolites. Halogenated alkenes,hydroquinones, and quinones have been shown to form toxic metabolitesvia the formation of S-conjugates with GSH. The kidney is the maintarget organ for compounds metabolized by this pathway. Selectivetoxicity to the kidney is the result of the kidney's ability toaccumulate intermediates formed by the processing of S-conjugates in theproximal tubular cells, and to bioactivate these intermediates to toxicmetabolites.

The administration of morphine and related compounds to rats and miceresults in a loss of up to approximately 50% of hepatic GSH. Morphine isknown to be biotransformed into morphinone, a highly hepatotoxiccompound, which is 9 times more toxic than morphine in mouse bysubcutaneous injection, by morphine 6-dehydrogenase activity. Morphinonepossesses an α,β-unsaturated ketone, which allows it to form aglutathione S-conjugate. The formation of this conjugate correlates withloss of cellular GSH. This pathway represents the main detoxificationprocess for morphine. Pretreatment with GSH protects againstmorphine-induced lethality in the mouse.

The deleterious effects of methylmercury on mouse neuroblastoma cellsare largely prevented by coadministration of GSH. GSH may complex withmethylmercury, prevent its transport into the cell, and increasecellular antioxidant capabilities to prevent cell damage. Methylmercuryis believed to exert its deleterious effects on cellular microtubulesvia oxidation of tubulin sulfhydryls, and by alterations due toperoxidative injury. GSH also protects against poisoning by other heavymetals such as nickel and cadmium.

Because of its known role in renal detoxification and its low toxicity,GSH has been explored as an adjunct therapy for patients undergoingcancer chemotherapy with nephrotoxic agents such as cisplatin, in orderto reduce systemic toxicity. In one study, GSH was administeredintravenously to patients with advanced neoplastic disease, in twodivided doses of 2,500 mg, shortly before and after doses ofcyclophosphamide. GSH was well-tolerated and did not produce unexpectedtoxicity. The lack of bladder damage, including microscopic hematuria,supports the protective role of this compound. Other studies have shownthat i.v. GSH coadministration with cisplatin and/or cyclophosphamidecombination therapy, reduces associated nephrotoxicity, while not undulyinterfering with the desired cytotoxic effect of these drugs.

Bohm, S., Battista-Spatti, G., DiRe, F., Oriana, S., Pilotti, S.,Tedeschi, M., Tognella, S. & Zunino, F.: A feasibility study ofcisplatin administration with low-volume hydration and glutathioneprotection in the treatment of ovarian carcinoma. Anticancer Res. 11:1613-1616. 1991.

Cozzaglio, L., Doci, R., Colla, G., Zunino, F., Casciarri, G. & Gennari,L.: A feasibility study of high-dose cisplatin and 5-fluorouracil withglutathione protection in the treatment of advanced colorectal cancer.Tumori 76: 590-594, 1990.

Di Re, F., Bohm, S., Oriana, S., Spatti, G.B., & Zunino, F.: Efficacyand safety of high-dose cisplatin and cyclophosphamide with glutathioneprotection in the treatment of bulky advanced epithelial ovarian cancer.Cancer Chemother. Pharmacol. 25: 355-360, 1990.

Nobile, M. T., Vidili, M. G., Benasso, M., Venturini, M., Tedeschi, M.,Zunino, F., & Rosso, R.: A preliminary clinical study ofcyclophosphamide with reduced glutathione as uroprotector. Tumori 75:257-258, 1989.

Clinical Use of Glutathione

Ten elderly patients with normal glucose tolerance and ten elderlypatients with impaired glucose tolerance (IGT) underwent GSH infusion,10 mg/min for 120 min, for a total dose of 1,200 mg in 2 hr, under basalconditions and during 75 g oral glucose tolerance tests and intravenousglucose tolerance tests. Basal plasma total glutathione levels wereessentially the same for normal and IGT groups, and GSH infusion underbasal conditions increased GSH to similar levels. This studydemonstrated that GSH significantly potentiated glucose-induced insulinsecretion in patients with IGT. No effect was seen on insulin clearanceand action.

The antihypertensive effect of an i.v. bolus of 1,844 mg. or 3,688 mg.GSH was studied in normal and mild to moderate essential hypertensivesubjects and in both hypertensive and non-hypertensive diabetics, bothtype I and type II. The administration of 1,844 mg. GSH produced a rapidand significant decrease in both systolic and diastolic blood pressure,within ten minutes, but which returned to baseline within 30 minutes, inboth groups of hypertensive patients and in non-hypertensive diabetics,but had no effect in normal healthy subjects. At the 3,699 mg. dose, notonly did the blood pressure decrease in the hypertensive subjects, butGSH produced a significant decrease in the blood pressure values innormal subjects as well.

GSH, 1,200 mg/day intravenously administered to chronic renal failurepatients on hemodialysis was found to significantly increase studiedhematologic parameters (hematocrit, hemoglobin, blood count) as comparedto baseline, and holds promise to reverse the anemia seen in thesepatients.

See, Costagliola, C., Romano, L., Scibelli, G., de Vincentiis, A.,Sorice, P. & DiBenidetto, A.: Anemia and chronic renal failure: atherapeutic approach by reduced glutathione parenteral administration.Nephron 61: 404-408, 1992.

Toxicological Effects of Glutathione.

The reported LD₅₀ of GSH in rats and mice via various routes ofadministration are listed in the table below. GSH has an extremely lowtoxicity, and oral LD₅₀ measurements are difficult to perform due to thesheer mass of GSH which has to be ingested by the animal in order to seeany toxic effects.

Route of Animal Admin. LD₅₀ Reference Mouse Oral 5000 mg/kg ModernPharmaceuticals of Japan, IV Edition. Tokyo, Japan Pharmaceutical,Medical and Dental Supply Exporters' Association, 1972, p 93. MouseIntraperitoneal 4020 mg/kg Modern Pharmaceuticals of Japan, IV Edition.Tokyo, Japan Pharmaceutical. Medical and Dental Supply Exporters'Association. 1972. p 93. Mouse Intraperitoneal 6815 mg/kg Toxicology,vol. 62. p. 205, 1990. Mouse Subcutaneous 5000 mg/kg ModernPharmaceuticals of Japan, IV Edition. Tokyo, Japan Pharmaceutical.Medical and Dental Supply Exporters' Association. 1972. p 93. MouseIntravenous 2238 mg/kg Japanese J. of Antibiotics, vol. 38. p. 137.1985. Mouse Intramuscular 4000 mg/kg Modern Pharmaceuticals of Japan,III Edition. Tokyo, Japan Pharmaceutical. Medical and Dental SupplyExporters' Association. 1968. p 97.

GSH can be toxic, especially in cases of ascorbate deficiency, and theseeffects may be demonstrated in, for example, ascorbate deficient guineapigs given 3.75 mmol/kg daily (1,152 mg/kg daily) in three divideddoses, whereas in non-ascorbate deficient animals, toxicity was not seenat this dose, but were seen at double this dose. See:

Dalhoff, K., Ranek, L., Mantoni, M. & Poulsen, H. E.: Glutathionetreatment of hepatocellular carcinoma. Liver 12: 341-343, 1992.

Dekant, W.: Bioactivation of nephrotoxins and renal carcinogens byglutathione S-conjugate formation. Toxicol. Letters 67: 151-60, 1993.

Domingo, J. L., Gomez, M., Llobet, J. M. & Corbella, J.: Chelatingagents in the treatment of acute vanadyl sulphate intoxication in mice.Toxicology 62: 203-211, 1990.

Martensson, J., Han, J., Griffith, O. W. & Meister, A.: Glutathioneester delays the onset of scurvy in ascorbate-deficient guinea pigs.Proc. Nat. Acad. Sci. USA 90: 317-321, 1993.

Thust, R, and Bach, B.: Exogenous glutathione induces sister chromatidexchanges, clastogenicity and endoreduplication in V79-E Chinese hamstercells. Cell Biol. Toxicol. 1: 123-31, 1985.

Aebi, S. & Lauterberg, B. H.: Divergent effects of intravenous GSH andcysteine on renal and hepatic GSH. Aer. J. Physiol. 263 (2 pt 2):R348-R352, 1992.

Ammon, H. P. T., Melien, M. C. M. & Verspohl, E. J.: Pharmacokinetics ofintravenously administered glutathione in the rat. J. Pharm. Pharmacol.38: 721-725, 1986.

Anderson, M. E., Powrie, F., Puri, R. N., & Meister, A.: Glutathionemonoethyl ester: Preparation, uptake by tissues, and conversion toglutathione. Arch. Biochem. Biophys. 239: 538-548, 1985.

Aw, T. Y., Wierzbicka, G. & Jones, D. P.: Oral glutathione increasestissue glutathione in vivo. Chem. Biol. Interact. 80: 89-97, 1991.

Borok, Z., Buhl, R., Grimes, G. J., Bokser, A. D., Hubbard, R. C.,Holroyd, K. J., Roum, J. H., Czerski, D. B., Cantin, A. M., & Crystal,R. G.: Effect of glutathione aerosol on oxidant-antioxidant imbalance inidiopathic pulmonary fibrosis. The Lancet 338: 215-216, 1991.

Buhl, R., Vogelmeier, C., Critenden, M., Hubbard, R. C., Hoyt, Jr., R.F., Wilson, E. M., Cantin, A. M. & Crystal, R. G.: Augmentation ofglutathione in the fluid lining the epithelium of the lower respiratorytract by directly administering glutathione aerosol. Proc. Natl. Acad.Sci. USA 87: 4063-4067, 1990.

Bump, E. A., al-Sarraf, R., Pierce, S. M. & Coleman, C. N.: Elevation ofmouse kidney thiol content following administration of glutathione.Radiother. Oncol. 23: 21-25, 1992.

Griffith, O. W., Bridges, R. J., & Meister, A.: Formation ofg-glutamyl-cyst(e)ine in vivo is catalyzed by γ-glutamyl transpeptidase.Proc. Natl. Acad. Sci. USA 78: 2777-2781, 1981.

Hagen, T. M., Wierzbicka, G. T., Bowman, B. B., Aw, T. Y. & Jones, D.P.: Fate of dietary glutathione. Disposition in the gastrointestinaltract. Am. J. Physiol. 259: G530-G535, 1990.

Hagen, T. M. & Jones, D. P.: Transepithelial transport of glutathione invascularly perfused small intestine of rat. Am. J. Physiol. 252:G607-G613, 1987.

Hagen, T. M., Wierzbicka, G. T., Sillau, A. H., Bowman, B. B. & Jones,D. P.: Bioavailability of dietary glutathione. Effect on plasmaconcentration. Am. J. Physiol. 259: G524-G529, 1990.

Hahn, R., Wendel, A. & Flohé, L.: The fate of extracellular glutathionein the rat. Biochim. Biophys. Acta 539: 324-337, 1978.

Puri, R. N., & Meister, A.: Transport of glutathione, asg-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Natl.Acad. Sci. USA 80: 5258-5260, 1983.

Viña, J., Perez, C., Furukawa, T., Palacin, M. & Viña, J. R.: Effect oforal glutathione on hepatic glutathione levels in rats and mice. Brit.J. Nutr. 62: 683-91, 1989.

Aebi, S., Asserto, R., & Lauterberg, B. H.: High-dose intravenousglutathione in man.: Pharmacokinetics and effects on cyst(e)ine levelsin plasma and urine. Eur. J. Clin. Invest. 21: 103-110, 1991.

Hagen, T. M. and Jones, D. P. Role of glutathione transport inextrahepatic detoxication, in Glutathione Centennial: MolecularPerspectives and Clinical Implications, N. Taniguchi, T. Higashi, Y.Sakamoto and A. Meister, eds. Acad. Press, New York, 1990.

Jones, D. P., Hagen, T. M., Weber, R., Wierzbicka, G. T., and Bonkovsky,H. L.: Oral administration of glutathione (GSH) increases plasma GSHconcentration in humans. FASEB J. 3: A1250 (5953), 1990.

Effects of Glutathione on the Circulatory System

Glutathione impacts many aspects of the circulatory system, includinginteractions with nitric oxide signaling, ischemia, and control overvascular endothelium.

Demopoulos, H. B., Flamm, E. S., Pietronigro, D. D., and Seligman, M.L.: Free radical pathology and antioxidants in regional cerebralischemia and central nervous system trauma. In: Anesthesia andNeurosurgery, eds. J. E. Cottrell and H. Tunndorf, C. V. Mosby, St.Louis, 1986, pp. 246-279.

Kagan, V. E., Bakalova, R. A., Koynova, G. M., Tyurin, V. A., Seriniva,E. A., Petkov, V. V., Staneva, D. S. and Packer, L.: Antioxidantprotection of the brain against oxidative stress. In: Free Radicals inthe Brain, eds. L. Packer, L. Prilipko, and Y. Christen.Springer-Verlag, New York, 1992, pp. 49-61.

Pietronigro, D. D., Demopoulos, H. B., Hovsepian, M. and Flamm, E. S.:Brain ascorbic acid depletion during cerebral ischemia. Stroke 13:117-119, 1982.

Shan, X., Aw, T. Y. and Jones, D. P.: Glutathione-dependent protectionagainst oxidative injury. Pharmac. Ther. 47: 61-71, 1990.

Simon, D. I., Stamler, J. S., Jaraki, O., et al.: Antiplateletproperties of protein S-nitrosothiols derived from nitric oxide andendothelium-derived relaxing factor. Arterioscler. Thromb. 13 (6):791-799, 1993.

Taccone-Gallucci, M., Lubrano, R., Clerico, A., Meloni, C., Morosetti,M., Meschini, L., Elli, M., Trapasso, E., Castello, M. A. & Casciani, C.U.: Administration of GSH has no influence on the RBC membrane:Oxidative damage to patients on hemodialysis. ASAIO Journal 38: 855-857,1992.

Use of High-Dose Oral GSH in Cancer Patients.

In one published study, eight patients with hepatocellular carcinomawere treated with 5 g oral reduced glutathione per day. Two patientswithdrew shortly after receiving GSH due to intolerable side-effects(gastrointestinal irritation and sulfur odor). The remaining patients,aged 27-63, three male and three female, did not experience side-effectsfrom this high dose of GSH and continued to take 5 g oral GSH forperiods ranging from 119 days (at which time the patient died from hertumor) to>820 days (this patient was still alive at the time ofpublication and was still taking 5 g oral GSH daily; his tumor had notprogressed and his general condition was good). Two of the femalepatients survived 1 year and exhibited regression or stagnation of theirtumor growth. The remaining two patients, both male, died as expectedwithin 6 months.

Experience in HIV-Infected Patients.

A commercially available nutritional formulation containing 3 grams ofreduced glutathione was given daily to a group of 46 AIDS patients for aperiod of three months by a group of private physicians. No significantGSH-related adverse effects were reported. No evidence of toxicitiesfrom laboratory studies or from clinical examinations was reported;however, no benefit was conclusively demonstrated.

Pharmacokinetics of Glutathione

The pharmacokinetics of intravenously administered GSH were determinedin the rat and interpreted by means of an open, two-compartment model.Following a bolus injection of 50-300 mmol/kg GSH, arterial plasmaconcentrations of (i) GSH, (ii) oxidized glutathione/GSSG, (iii) totalthiols, and (iv) soluble thiols minus GSH, were elevated and thenrapidly decreased non-exponentially, as anticipated. With increasingdose, the rate constant for drug elimination and plasma clearanceincreased form 0.84 to 2.44 mL/min. and the half-life of the eliminationphase decreased from 52.4 to 11.4 minutes. Both the apparent volume ofdistribution and the degree of penetration of GSH into the tissues werediminished with increasing dose (from 3.78 to 1.33 L/Kg and from 6.0 to0.51 as k₁₂/k₂₁, respectively). The data indicate that GSH is rapidlyeliminated. This is mainly due to rapid oxidation in plasma rather thanby increased tissue extraction or volume distribution. Thus, plasma GSHlevels appear to be quickly regulated by which the body may maintainconcentrations within narrow physiological limits.

When single doses of 600 mg GSH were administered intravenously tosheep, GSH levels in venous plasma and lung lymph rose transiently. Themean concentration was approximately 50 mM for venous plasma, peaking at30 min, and returning to baseline after 45 minutes. Lung lymph peaklevel was about 100 mM at 15 min, returning to baseline after 30minutes. Average epithelial lining fluid (ELF) levels were variable butshowed no significant increase over baseline during the three hourobservation period. Urine excretion was rapid with peak levels at 15minutes. In both plasma and lung lymph, GSH accounted for greater than95% of the total glutathione (GSH plus GSSG). In ELF 75.4% of thebaseline glutathione was in the reduced form, whereas in urine 59.6% waspresent as GSH.

Orally ingested reduced glutathione is absorbed intact from the smallintestine in a rat model, specifically in the upper jejunum. It is notedthat rat metabolism differs from man, and therefore the results of ratstudies should be verified in man before the results are extrapolated.Plasma GSH concentration in rats increased from 15 to 30 mM afteradministration of GSH either as a liquid bolus (30 mM) or mixed (2.5-50mg/g) in AIN-70 semi-synthetic diet (11). GSH concentration was maximalat 90-120 minutes after GSH administration and remained high for over 3hours. Administration of the amino acid precursors of GSH had little orno effect on plasma GSH values, indicating that GSH catabolism andre-synthesis do not account for the increased GSH concentration seen.Inhibition of GSH synthesis and degradation byL-buthionine-[S,R]-sulfoximine (BSO) and acivicin showed that theincreased plasma GSH came mostly from absorption of intact GSH insteadof from its metabolism. Plasma protein-bound GSH also increased afterGSH administration, with a time course similar to that observed for freeplasma GSH. Thus, dietary GSH can be absorbed intact and results in asubstantial increase in blood plasma GSH.

Administration of oral GSH increased hepatic glutathione levels in: (i)rats fasted 48 hours, (ii) mice treated with GSH depletors, and (iii)mice treated with paracetamol (a drug which promotes a depletion ofhepatic GSH followed by hepatic centrilobular necrosis). In theseexperiments, the animals were orally intubated with 1000 mg/kg bodyweight GSH. Mean pretreatment values in 48-hour fasted rats were 3.0-3.1mmol/g fresh hepatic tissue. Mean values after treatment were 5.8, 4.2,and 7.0 mmol/g fresh hepatic tissue for 2.5, 10, and 24 hourspost-treatment, respectively. Mice were given an oral dose of GSH (100mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min inblood plasma and after 1 hr in liver, kidney, heart, lung, brain, smallintestine and skin. GSH concentrations in plasma increased from 30 mM to75 mM within 30 min of oral GSH administration, consistent with a rapidflux of GSH from the intestinal lumen to plasma. No increases overcontrol values were obtained in most tissues except lung over the sametime course. Mice pretreated with the GSH synthesis inhibitor BSO hadsubstantially decreased tissue concentrations of GSH, and oraladministration of GSH to these animals resulted instatistically-significant increases in the GSH concentrations of kidney,heart, lung, brain, small intestine and skin but not in liver.

Fahey, R. C., and Newton, G. L.: Determination of low molecular weightthiols using monobromobimane fluorescent labeling and high-performanceliquid chromatography. Meth. Enzymol. 143: 85-96, 1987. See:

Mills, B. J., Richie, J. P. Jr., and Lang, C. A.: Sample processingalters glutathione and cysteine values in blood. Anal. Biochem. 184:263-267, 1990.

Richie, J. P. Jr., and Lang, C. A.: The determination of glutathione,cyst(e)ine, and other thiols and disulfides in biological samples usinghigh-performance liquid chromatography with dual electrochemicaldetection. Anal. Biochem. 163: 9-15, 1987.

Tietz, F.: Enzymic method for quantitative determination of nanogramamounts of total and oxidized glutathione: Applications to mammalianblood and other tissues. Anal. Biochem. 27: 502-22, 1969.

The kinetics and the effect of glutathione on plasma and urinesulphydryls were studied in ten healthy human volunteers. Following theintravenous infusion of 2000 mg/m² of GSH the concentration of totalglutathione in plasma increased from 17.5-13.4 mmol/Liter (mean=/−SD) to823-326 mmol/Liter. The volume of distribution of exogenous glutathionewas 176-107 Ml/Kg and the elimination rate constant was0.063-0.027/minute, corresponding to a half-life of 14.1-9.2 minutes.Cysteine in plasma increased from 8.9-3.5 mmol/Liter to 114-45mmol/Liter after the infusion. In spite of the increase in cysteine, theplasma concentration of total cyst(e)ine (i.e. cysteine, cystine, andmixed disulphides) decreased, suggesting an increased uptake of cysteinefrom plasma into cells. The urinary excretion of glutathione and ofcyst(e)ine was increased 300-fold and 10-fold respectively, in the 90minutes following the infusion.

Normal healthy volunteers were given an oral dose of GSH to determinewhether dietary GSH could raise plasma GSH levels. Results showed thatan oral dose of GSH (15 mg/kg) raised plasma glutathione levels inhumans 1.5-10 fold over the basal concentration in four out of fivesubjects tested, with a mean value three times that of normal plasma GSHlevels. Plasma GSH became maximal 1 hour after oral administration,dropping to approximately ½ maximal values after three hours. Equivalentamounts of GSH amino acid constituents failed to increase plasma levelsof GSH. GSH bound to plasma proteins also increased with the same timecourse as seen with free GSH.

SUMMARY OF THE INVENTION

The present inventors have found that oral glutathione bioavailabilityand efficiency may be increased by the administration ofpharmaceutically stabilized reduced glutathione in a bolus on an emptystomach.

The present inventors have also found that glutathione is efficientlyabsorbed from mucous membranes, especially the sublingual mucosa andlumen of the duodenum and initial part of the ileum.

One aspect of the present invention embodies the use of glutathioneadministered pharmacologically, to alter a redox state within the cellsof an organism, and to therefore alter an expression of redox-dependentfactors, such as NF-κB and PEDF.

Therefore, e.g., as a result of bioavailable administration ofglutathione (GSH), the redox balance of the tissues will be shiftedtoward the reduced state. This is especially the case in the event oftissues with a high or abnormally high metabolic demand, wherein aproduction of free radicals is excessive. In that case, the presence ofpharmacologically administered reduced glutathione will be expected tohave an even greater impact in altering a redox balance in the cells.Thus, it is believed that the influence of exogenous glutathione will beparticularly seen in proximity to those tissues that are at risk ofischemia.

It is noted that glutathione's effects are not limited to increasing orsustaining levels of PEDF, but rather the action of glutathione may beexerted on many different tissues and cell functions. It is particularlynoted that glutathione regulated redox state may control cell functionthrough gene induction, transcriptional, translational,posttranslational, or receptor-mediated effects, on a variety offactors.

In the case of PEDF, the administration of glutathione would be expectedto act as an antineoplastic therapy by (a) reducing neovascularization,(b) serving as an influence toward differentiated states of cells, and(c) supporting the normal function of tissues, such as neurons. It isparticularly noted that, in this respect, the action of glutathione asan antioxidant and free radical scavenger is believed to be distinct andseparate.

In the case of NF-κB, glutathione administration would be expected toforestall the cascade which activates certain viral replication,including HIV.

Glutathione may also alleviate certain immune and autoimmune disorders,including rheumatoid arthritis, and alter glucocorticoid effects.

It is thought that transplantation of neurons (or their precursor cells)may cure or alleviate certain pathologies. For example, in Parkinson'sdisease, transplantation of specific fetal brain cells into patientscould alleviate or cure certain problems associated with the disease.However, the transplanted cells would have to appropriatelydiffereniate, and remain differentiated, in situ to functionally replacethe pathological or dead cells. This involves creating and maintaining amicroenvironment for the cells having the appropriate growth factors andstimuli. The maintenance of a high concentration of reduced glutathionecould promote, for example, the secretion of PEDF by the astroglia, orassist genetically modified (transfected) astroglia to produce highlevels of PEDF, thus providing an environment rich in neural growthfactors.

Ischemia Reperfusuion injury is also a particular concern intransplantation, and the pretreatment of the cells with relatively highlevels of glutathione may reduce the free radical damage to the cells aswell as the levels of secondary redox messengers.

As used herein, the term “pharmaceutically stabilized glutathione”refers to glutathione which is maintained in a reduced form withoutsubstantial cyclization. This stabilization may be effected by theaddition of one or more agents that, together with the glutathione,provide a pharmaceutical formulation which is capable of deliveringnative reduced glutathione.

The present invention also includes novel combinations of glutathioneand other pharmacological agents and in novel dosage forms and means foradministration.

The oral administration of pharmaceutically stabilized reducedglutathione, presented as a charge transfer complex in relatively highconcentration may produce a significant, predictable increase inintracellular glutathione levels in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is shown by way of example in the drawings, in which:

FIG. 1 shows a graph of a clinical response of an HIV infected subjectto 1 gram of administered glutathione; and

FIG. 2 shows a table of clinical study results.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been found that, in otherwise healthy HIV infected humans, theintracellular glutathione levels in the peripheral blood mononucleocytes(PBMs) was significantly increased after oral administration ofstabilized glutathione. This is achieved by providing a glutathioneformulation which ensures delivery of adequate dose of pharmaceuticallystabilized, reduced glutathione, with rapid dissolution before theduodenum. The formulation is administered to efficiently provide a highconcentration of glutathione in the duodenum, i.e., on an empty stomach,to enhance uptake.

A preferred formulation includes 250 mg, or more of reduced glutathionewith at least equimolar ascorbic acid, to fulfill three functions: actsas a sacrificial non-specific antioxidant during preparation, storageand after ingestion; reduces or neutralizes static electrical charge ofglutathione powder, allowing dense packing of capsules; and acts as alubricant for the encapsulation device. The ascorbic acid also maintainsan acidic and reducing environment, which pharmaceutically stabilizesthe glutathione molecule. Ascorbic acid is believed to form a chargecouple with glutathione which enhances penetration through cellmembranes, and reduces the tendency for the gamma-glutamyl and glycinylresidues to assume a cyclic conformation or to form an internal cyclicamide. The ascorbate thus complexes with the glutathione in solution tomaintain a linear conformation. This linear conformation, in turn,stericly hinders the free cysteinyl thiol group. This steric hindrancestabilizes a free radical that may be formed, and thus maintains thebiological activity of glutathione.

A cyclic form of glutathione, which may occur under certain conditions,such as neutral to basic pH, exposes the sulfhydryl moiety, making itmore reactive. Under alkaline pH, cyclic amide formation is promoted,leaving a potentially toxic compound. The cyclic glutathione compositionis a potential structural analog that may inhibit glutathione reductase,glutathione peroxidase and specific glutathione transporter proteins.

Likewise, oxidizing conditions promote disulfide formation (GSSG andPr-S-S-G), which may reduce bioavailability of glutathione andcounteract some of the potential benefits of glutathione administration.Further, oxidizing conditions also promote desulfuration, resulting inopthalmic acid formation (or other compounds), which may be toxic orinhibit efficient glutathione absorption.

A preferred oral formulation thus preferably includes an effectiveamount of glutathione mixed with a stabilizing agent, which isadministered under such conditions that the concentration of glutathioneattained in the lumen of the latter portion of the duodenum is higherthan the plasma glutathione concentration, and preferably higher thanthe intercellular concentration of the epithelial lining cells. Thus,for example, a glutathione and ascorbic acid capsule is taken on anempty stomach. The reducing agent, preferably ascorbic acid, preventsoxidation of the glutathione during packaging and storage, and furthermay stabilize the glutathione in the relatively alkaline conditions ofthe duodenum prior to absorption. Desulfuration of glutathione leads tothe formation of ophthalmic acid, the serine analog of glutathione,which inhibits glutathione uptake. This protocol is in contrast to priorart administration methods, which direct taking glutathione capsulesafter meals. By diluting glutathione with food, degradative enzymes arediluted and alkaline conditions buffered; however, according to thepresent invention, the rapidity of absorption allows highbioavailability with only a small amount of degradation.

The present invention also advantageously provides a method of use andpharmaceutical formulation of glutathione combined and anotherpharmaceutically active composition, wherein the other composition isselected from a broad group consisting of:

easily oxidized compositions,

antioxidant compositions,

compositions with oxidant effects,

compositions for the treatment of pathology associated with:

suppressed total glutathione levels,

suppressed reduced glutathione levels,

relatively oxidized conditions in the organism,

uncontrolled free radical or oxidizing reactions, or

conditions where a more reduced state is desirable.

Glutathione may be used alone or in combination with other knowncompositions for the treatment or palliation of AIDS, HIV infection orretroviral replication (e.g., HTLV I, HTLV-II, HTLV-III, etc.), herpesvirus replication (e.g., Herpes simplex type I, Herpes simplex type II.Herpes zoster (varicella), CMV, EBV, HHV-8, etc.), rabies, ebola virus,influenza virus, CHF, coronary artery disease, status post coronaryartery restenosis, Diabetes mellitus, Macular Degeneration, and/orhepatitis (toxic or infectious). In addition, certain neurologicalconditions, such as amyotrophic lateral sclerosis, Parkinson's disease,Alzheimer's disease and others may also benefit from antioxidanttherapies. Further, a number of pharmaceutical therapies, especiallythose that cross the blood brain barrier, are associated with sideeffects that relate to oxidative effects. Other drugs, such asTamoxifen, are associated with macular degeneration. Thus, glutathionemay be administered in accordance with the present invention to treatviral or certain bacterial infections, chronic diseases, detoxify drugs,treat or alleviate oxidative and lipid peroxidative disorders, and toreduce the long-term effects of oxidant agents, such as superoxide,which include carcinogenesis and aging.

It is noted that in the case of diseases which have as a part of theiretiology a precipitation of proteins, such as amyloid diseases, e.g.,Alzheimer's disease, the alteration of redox potential of the medium mayhave a dramatic effect on protein solubility. Thus, as the mediumbecomes more oxidized, the proteins will typically have more disulfidelinkages, both defining the secondary structure of the peptide, andpotentially forming cross linkages with other moieties. On the otherhand, the administration of reduced glutathione will result in areducing environment, with correspondingly more free sulfhydryl groups.Therefore, it is expected that administration of glutathione willprovide an effective treatment, or part of a treatment regimen, for suchdiseases. It is also noted that precipitated peptides may be involved infree radical reactions, which will also be countered by glutathioneadministration.

Glutathione may also be used, alone or in combination with othertherapies for the treatment of free radical associated neurologicalconditions, for example, Alzheimer's disease, Parkinson's disease,catecholamine toxicities, other free-radical associated toxicities,stroke and transient ischemic events, spinal chord injury and othertraumatic injuries to nerve tissue, peripheral neuropathies, possiblyprion-associated illness, infectious agent pathology and inflammatorypathology, or to reduce the free-radical associated toxicity of drugsadministered to treat these conditions.

Mycoplasma infections, such as mycoplasma pneumonia, are believed tocause pathology due to free radical reactions within cells by theseintracellular parasites. Therefore, glutathione may be administeredalone or in combination with an anti-mycoplasma antibiotic for thetreatment of mycoplasma infections.

The present invention may also be used to increase or supplement theglutathione levels in normal mammals. This may be desired, for example,for prophylaxis against ischemic events, free radical damage from sun,chemicals, or other environmental exposure, and to reduce a cancer risk.

In fact, since oxidizing conditions in an organism are generallyundesirable, and where necessary the mechanisms for producing oxidizingconditions typically overpower ingested antioxidants, a large number ofmedications and drugs are appropriate for combination with glutathione.However, certain conditions may require care in the administration ofglutathione. Further, certain cancer chemotherapy regimens rely onexhaustion of cellular free radical quenching mechanisms to selectivelykill target cells. Finally, cellular apoptosis, or programmed celldeath, relies on exhaustion of reduced glutathione levels in cells(mitochondria), resulting in death. Where this mechanism is required orphysiologically correct, interruption by exogenous glutathione may beundesirable. Further, glutathione may interact with some compositions,either to non-specifically reduce or combine with the chemical moiety,or to alter a metabolism after ingestion; unless accounted for, theseeffects may be undesirable.

Glutathione may have efficacy in treatment of male infertility. Thus,glutathione may remedy mitochondrial defects or deficiency. Lenzi, A.,Lombardo, F., Gandini, L., Culasso, F. & Dondero, F.: Glutathionetherapy for male infertility. Arch. Androl. 29: 65-68, 1992.

A known anti-HIV therapy, 3′-azidothymidine (zidovudine, AZT), acts as apotent reverse transcriptase inhibitor. This drug, however, generatesfree radicals and is toxic to mitochondria, and is associated with amyopathy. Glutathione may therefore be administered in conjunction withAZT to reduce toxicity while not interfering with the reversetranscriptase inhibitory activity, thus increasing the therapeuticindex. Likewise, glutathione may also be used to increase thetherapeutic index of other drugs that have a significant free-radicalassociated toxicity.

There are a number of conditions which are believed to be associatedwith reduced intracellular antioxidant levels, including AIDS, diabetes,macular degeneration, congestive heart failure, vascular disease andcoronary artery restenosis, Herpes virus infection, toxic and infectioushepatitis, and rabies. Certain interstitial lung disease may be due toinsufficient glutathione levels. Further, various toxins and medicationsmay also result in free radical reactions, including types of cancerchemotherapy. Therefore, the present invention holds potential to treatthese diseases and conditions by the use of a convenient, effective oralformulation of glutathione. Thus, the administration of exogenousglutathione supplements the hepatic output to help maintain reducedconditions within the organism. As noted above, the failure to quenchfree radical reactions allows an undesirable cascade resulting in damageto macromolecules, lipid peroxidation, and generation of toxiccompounds. The maintenance of adequate glutathione levels is necessaryto block these free radical reactions.

Glutathione also has the ability to form complexes with metals. Forexample, as discussed above, glutathione forms chelation complexes withnickel, lead, cadmium, mercury, vanadium and manganese. Glutathione alsoforms circulating complexes with copper in the plasma. According to thepresent invention, glutathione may be administered to treat metaltoxicity. It is believed that the glutathione-metal complexes will beexcreted, thus reducing the metal load. Thus, glutathione may beadministered for the treatment of toxicity associated with iron, copper,nickel, lead, cadmium, mercury, vanadium, manganese, cobalt, transuranicmetals, such as plutonium, uranium, polonium, and the like. As comparedto EDTA, glutathione has a reduced tendency to chelate calcium,providing a significant advantage. It is noted that the chelationproperties of glutathione are separate from the antioxidant properties;however, some metal toxicities are free radical mediated, for exampleiron, and therefore glutathione administration for these conditions isparticularly advantageous.

In order to provide high bioavailability, it has been found desirable toprovide a relatively high concentration of reduced glutathione inproximity to the mucous membrane, e.g., the duodenum for oraladministration. Thus, in contrast to prior methods, the glutathione ispreferably administered as a single bolus on an empty stomach. Thepreferred dosage is between about 100-10,000 mg. glutathione, and morepreferably between about 250-3,000 mg. glutathione. Further, theglutathione formulation is preferably stabilized with a reducing agent(antioxidant), preferably ascorbic acid, to reduce oxidation both duringstorage and in the digestive tract prior to absorption. The use ofcrystalline ascorbic acid has the added benefit of reducing the staticcharge of glutathione for improved encapsulation and serving as alubricant for the encapsulation apparatus. However, other staticdissipation methods or additives may be employed, and other antioxidantsmay be employed. The preferred dosage form is a capsule, e.g., atwo-part gelatin capsule, which protects the glutathione from air andmoisture, while dissolving quickly in the stomach.

The digestive tract is believed to have specific facilitated or activetransport carriers for glutathione, which allow uptake of glutathionefrom the intestinal lumen without degradation. According to the presentinvention, the uptake through this mechanism is maximized by providing ahigh concentration gradient and avoiding the presence or production oftransport inhibitors, such as ophthalmic acid. Thus, the preferredmethod of oral administration according to the present invention employsan uptake mechanism that differs from glutathione administed using priormethods, as well as most other thiol compounds.

The oral mucosa have been found to allow rapid and efficient uptake ofglutathione into the blood. In contrast to the digestive tract, thesignificance of facilitated or active transport mechanisms in the oralmucosa is believed to be low; rather, a high concentration ofglutathione in the oral mucosa is believed to permit passive transportof the glutathione through the cells or around the cells into thecapillary circulation. Therefore, compositions intended for absorptionthrough the oral mucosa, e.g., for sublingual administration, arepreferably of high purity, as contaminants may be absorbed similarly toglutathione, and as relatively small, uncharged molecules. Therefore,the composition preferably includes ascorbic acid that helps to maintainthe glutathione in a reduced state, maintains a somewhat acidicenvironment in the mouth to avoid deprotonation of the glutamic acidresidue, without causing substantially all of the amines to beprotonated.

It has been found, contrary to reports of other scientists, thatglutathione is not substantially degraded in the stomach, and therefore,the release of the glutathione need not diluted in the stomach orrelease be delayed. In fact, according to the present invention, theglutathione formulation is preferably released and dissolved in thestomach. The addition of stabilizer, i.e., ascorbic acid, furtherimproves the ability of the glutathione to reach its site of absorptionin the intestine undegraded. Once past the stomach, it is important thatthe glutathione be immediately available for absorption, as thedesulfurases and peptidases from the pancreas, as well as the increasein pH, do tend to degrade the glutathione. The desulfurase producesophthalmic acid, which interferes with glutathione absorption. Thus, byproviding a high concentration of glutathione in the duodenum, withoutsubstantial dilution, the glutathione may be absorbed at a maximum rate.While the degradation of glutathione in the latter part of the duodenumand ileum may compete with the absorption process, the present methodprovides significant bioavailability. In fact, studies have demonstratedabout 90% bioavailability of orally administered glutathione accordingto the present invention.

The capsule is preferably a standard two-part hard gelatin capsule ofdouble-O (OO) size, which may be obtained from a number of sources.After filling, the capsules are preferably stored under nitrogen, toreduce oxidation during storage. The capsules are preferably filledaccording to the method of U.S. Pat. No. 5,204,114, incorporated hereinby reference in its entirety, using crystalline ascorbic acid as both anantistatic agent and stabilizer. Further, each capsule preferablycontains 500 mg of glutathione and 250 mg of crystalline ascorbic acid.A preferred composition includes no other excipients or fillers;however, other compatible fillers or excipients may be added. Whilediffering amounts and ratios of glutathione and stabilizer may be used,these amounts are preferable because they fill a standard double-Ocapsule, and provide an effective stabilization and high dose. Further,the addition of calcium carbonate, a component of known high doseglutathione capsules, is avoided as there may be impurities in thiscomponent. Further, calcium carbonate acts as a base, neutralizingstomach acid, which accelerates degradation of glutathione in the smallintestine.

Because the glutathione and ascorbic acid are administered in relativelyhigh doses, it is preferred that these components be highly purified, toeliminate impurities, toxins or other chemicals, which may destabilizethe formulation or produce toxic effects or side effects. As statedabove, the formulation may also include other pharmaceutical agents, ofvarious classes.

Glutathione is advantageously administered over extended periods.Therefore, one set of preferred useful combinations include glutathioneand drugs intended to treat chronic conditions which are well absorbedon an empty stomach, and do not have adverse interactions or reduced orvariable combined absorption.

One particular class of drugs includes central or peripheral adrenergicor catecholenergic agonists, or reuptake blockers, which may produce anumber of toxic effects, including neurotoxicity, cardiomyopathy andother organ damage. These drugs are used, for example, as cardiac,circulatory and pulmonary medications, anesthetics andpsychotropic/antipsychotic agents. Some of these drugs also have abusepotential, as stimulants, hallucinogens, and other types ofpsychomimetics. Other free radical initiation associated drugs includethorazine, tricyclic antidipressants, quinolone antibiotics,benzodiazepines, acetaminphen and alcohol.

Therefore, it is an aspect of the present invention to provide an oralpharmaceutical formulation comprising glutathione in an amount ofbetween about 50-10,000 mg, and an effective amount of a pharmacologicalagent capable of initiating free radical reactions in a mammal. Thepharmacological agent is, for example, an adrenergic, dopaminergic,serotonergic, histaminergic, cholinergic, gabaergic, psychomimetic,quinone, quinolone, tricyclic, and/or steroid agent.

Hepatic glutathione is consumed in the metabolism, catabolism and/orexcretion of a number of agents. The depletion of hepatic glutathionemay result in hepatic damage or a toxic hepatitis. Such agents mayinclude, for example, aminoglycoside antibiotics, acetominophen,morphine and other opiates. High dose niacin, used to treathypercholesterolemia, has also been associated with a toxic hepatitis.The present invention therefore encompasses an oral pharmaceuticalformulation comprising glutathione in an amount of between about50-10,000 mg, administered in conjunction with and an effective amountof a pharmacological agent that consumes hepatic glutathione reserves.

A number of pathological conditions result in hepatic damage. Thisdamage, in turn, reduces the hepatic reserves of glutathione and theability of the liver to convert oxidized glutathione to its reducedform. Other pathological conditions are associated with impairedglutathione metabolism. These conditions include both infectious andtoxic hepatitis, cirrhosis, hepatic primary and metastatic carcinomas,traumatic and iatrogenic hepatic damage or resection. The presentinvention encompasses a pharmaceutical formulation comprisingglutathione and an antiviral or antineoplastic agent. The antiviral orantineoplastic agent is, for example, a nucleoside analog.

Glutathione is broken down, and cysteine excreted, possibly in theurine. Very high doses of glutathione may therefore result incysteinuria, which may result in cysteine stones. Other long termtoxicity or adverse actions may result. Therefore, a daily intake ofgreater than about 10 gm. for extended period should be medicallymonitored. On the other hand, individual doses below about 50 mg. areinsufficient to raise the concentration of the duodenal lumen to highlevels to produce high levels of absorption, and to provide clinicalbenefit. Therefore, the preferred formulations according to the presentinvention have glutathione content greater than 50 mg, and provided inone or more doses totaling up to about 10,000 mg per day.

In the treatment of HIV infection, it is believed that the oraladministration of a relatively high dose bolus of glutathione, i.e., 1-3grams per day, on an empty stomach, will have two beneficial effects.First, HIV infection is associated with a reduction in intracellularglutathione levels in PBMs, lung, and other tissues. It is furtherbelieved that by increasing the intracellular glutathione levels, thefunctioning of these cells may be returned to normal. Therefore, theadministration of glutathione according to the present invention willtreat the effects of HIV infection. Therefore, the present inventionencompasses the oral administration of glutathione and ascorbic acid,optionally in combination with an antiretroviral agent. It is noted thatthe transcription mechanisms and control involved in retroviralinfection is believed to be relatively conserved between various types.Therefore, late stage retroviral suppression is expected for the varioustypes of human retroviruses and analogous animal retroviruses.

Second, it has been found in in vitro tests that by increasing theintracellular levels of glutathione in infected monocytes to the highend of the normal range, the production of HIV from these cells may besuppressed for about 35 days. This is believed to be related to theinterference in activation of cellular transcription by cytokines,including NF-κB and TNF-α. Therefore, the infectivity of HIV infectedpersons may be reduced, helping to prevent transmission. This reductionin viral load may also allow the continued existence of uninfected butsusceptible cells in the body.

Glutathione, administered according to the present method, is believedto be effective in the treatment of congestive heart failure (CHF). InCHF, there are believed to be two defects. First, the heart muscle isweakened, causing enlargement of the heart. Second, peripheral vasospasmis believed to be present, causing increased peripheral resistance.Glutathione is effective in enhancing the effects of nitric oxide, andtherefore is believed to be of benefit to these patients by decreasingvasoconstriction and peripheral vascular resistance, while increasingblood flow to the tissues. While nitroso-glutathione is more effectiveat preventing platelet aggregation than at vasodilation, it isnevertheless a potent vasodilator with a longer half-life than nitricoxide alone. Further, since a relative hypoxia of the tissues isassociated with free radical-mediated cellular damage, the presence ofglutathione will also help to block this damage. The present inventiontherefore encompasses the oral administration of glutathione inconjunction with a congestive heart failure medication, for example,digitalis glycosides, dopamine, methyldopa, phenoxybenzamine,dobutamine, terbutaline, amrinone, isoproterenol, beta blockers, calciumchannel blockers, such as verapamil, propranolol, nadolol, timolol,pindolol, alprenolol, oxprenolol, sotalol, metoprolol, atenolol,acebutolol, bevantolol, tolamolol, labetalol, diltiazem, dipyridamole,bretylium, phenytoin, quinidine, clonidine, procainamide, acecainide,amiodarione, disopyramide, encainide, flecanide, lorcainide, mexiletine,tocainide, captopril, minoxodil, nifedipine, albuterol, pargyline,vasodilators, including nitroprusside, nitroglycerin, phentolamine,phenoxybenzamine, hydrazaline, prazosin, trimazosin, tolazoline,trimazosin, isosorbide dinitrate, erythrityl tetranitrate, asprin,papaverine, cyclandelate, isoxsuprine, niacin, nicotinyl alcohol,nylidrin, diuretics, including furosemide, ethacrynic acid,spironolactone, triamterine, amiloride, thiazides, bumetanide, caffeine,theophylline, nicotine, captopril, salalasin, and potassium salts.

It has been found that elevated levels of homocysteine as a significantrisk in vascular disease, such as atherosclerosis, venous thrombosis,heart attack and stroke, as well as neural tube defects and neoplasia.Moghadasian et al., “Homocyst(e)ine and Coronary Artery Disease”, Arch,Int. Med. 157 (10): 2299-2308 (Nov. 10, 1997), incorporated herein byreference. Homocystiene promotes free radical reactions. In patientswith defective homocysteine metabolism, relatively high levels ofhomocysteine are present in the blood. According to the presentinvention, glutathione is administered to patients with elevatedhomocysteine levels.

It was believed that, because hepatocytes produce reduced glutathionethrough a feedback-inhibited pathway, hepatocytes would not effectivelyabsorb reduced glutathione from the plasma. Therefore, an insult tohepatocytes, for example from toxic or infectious origin, whichotherwise suppressed glutathione production, would result in cellulardamage or death. In fact, the present inventors believe that this is notthe case, at least in the case of compromised hepatocytes. Therefore, itis an aspect according to the present invention to treat hepatitis, ofvarious types, with orally administed glutathione. For example, bothalcohol and acetaminophen are both hepatotoxic, and result in reducedhepatocyte glutathione levels. Therefore, these toxicities may betreated according to the present invention. Glutathione may also beeffective in the treatment of other types of toxicities, to variouscells or organs, which result in free radical damage to cells or reducedglutathione levels. Hepatitis may also have viral etiology, and the useof glutathione may be beneficial in a similar manner to the use ofglutathione in the treatment of mangement of HIV infection. Theglutathione may act to reduce expression of viral genes, as well asreduce the oxidative challenge resulting from active viral replication.Glutathione may also reduce viral disulfide bonds, reducing viralinfectivity.

Diabetes, especially uncontrolled diabetes, results in glycosylation ofvarious enzymes and proteins, which may impair their function orcontrol. In particular, the enzymes which produce reduced glutathione(e.g., glutathione reductase) become glycosylated and non-functional.Therefore, diabetes is associated with reduced glutathione levels, andin fact, many of the secondary symptoms of diabetes may be attributed toglutathione metabolism defects. The present invention may therefore beapplied to supplement diabetic patients with glutathione in order toprevent the major secondary pathology. The present invention alsoencompasses an oral pharmaceutical formulation comprising glutathioneand an antihyperglycemic agent.

Glutathione, due to its strong reducing potential, breaks disulfidebonds. It is believed that most normal proteins are not denatured, to agreat extent, by normal or superphysiologic levels of glutathione. It isbelieved, however, that opiate receptors are deactivated by high normallevels of glutathione. It is therefore believed that glutathioneadministration may be of benefit for the treatment of obesity and/oreating disorders, other addictive or compulsive disorders, includingtobacco (nicotine) and opiate additions.

The present invention also encompasses the administration of glutathionein conjunction with nicotine. The physiologic effects of nicotine arewell known. Glutathione, due to its vasodilatory effects, improvescerebral blood flow, resulting in a synergistic cerebralfunction-enhancing effect.

In mammals, the levels of glutathione in the plasma are relatively low,in the micromolar range, while intracellular levels are typically in themillimolar range. Therefore, the intracellular cytosol proteins aresubjected to vastly higher concentrations of glutathione thanextracellular proteins. The endoplasmic reticulum, a cellular organelle,is involved in processing proteins for export from the cell. It has beenfound that the endoplasmic reticulum forms a separate cellularcompartment from the cytosol, having a relatively oxidized state ascompared to the cytosol, and thereby promoting the formation ofdisulfide links in proteins, which are often necessary for normalactivity. Hwang, C., et al., “Oxidized Redox State of Glutathione in theEndoplasmic Reticulum”, Science 257: 1496-1502 (Sep. 11, 1992),incorporated herein be reference. In a number of pathological states,cells may be induced to produce proteins for export from the cells, andthe progression of the pathology interrupted by interference with theproduction and export of these proteins. For example, many viralinfections rely on cellular production of viral proteins forinfectivity. Interruption of the production of these proteins willinterfere with infectivity. Likewise, certain conditions involvespecific cell-surface receptors, which must be present and functional.In both these cases, cells that are induced to produce these proteinswill deplete reduced glutathione in the endoplasmic reticulum. It isnoted that cells that consume glutathione (GSH) will tend to absorbglutathione from the plasma, and may be limited by the amounts present.Therefore, by increasing plasma glutathione levels, even transiently,the reducing conditions in the endoplasmic reticulum may be interferedwith, and the protein production blocked. Nornal cells may also besubjected to some interference; however, in viral infected cells, orcells abnormally stimulated, the normal regulatory mechanisms may not beintact, and the redox conditions in the endoplasmic reticulum controlledby the availability of extracellular glutathione. In these conditions,the pharmaceutical administration of glutathione may produce significanteffects.

Reproduction of herpes viruses, which are DNA viruses, is inhibited orreduced in cell culture by the administration of extracellularglutathione. Therefore, according to the present invention, herpes virusinfections may be treated by administering glutathione according to thepresent invention. The known herpes viruses include herpes simplex virusI, herpes simplex virus II, herpes zoster, cytomegalovirus, Epstein Barrvirus, as well as a number of other known viruses.

It is also believed that infection by the rabies virus, an RNA virus,may be treated by the administration of glutathione. While standardtreatments are available, and indeed effective when timely administered,glutathione may be useful in certain circumstances. Therefore, rabiesvirus infection may be treated, at least in part, according to thepresent invention. One available treatment for rabies is an immuneserum. The present invention therefore encompasses the parenteraladministration of glutathione in combination with an antibody.Glutathione may also be administered separately.

Coronary heart disease risk is increased by the consumption of ahigh-fat diet, and reduced by the intake of antioxidant vitamins,including vitamin E and vitamin C, as well as flavonoids. High fat mealsimpair the endothelial function through oxidative stress, resulting inimpaired nitric oxide availability. It has been found that vitamin C andvitamin E restores the vasoconstriction resulting from nitric oxideproduction by endothelium after a high fat meal. Plotnick, G. D. et al.,“Effect of Antioxidant Vitamins on the Transient Impairment ofEndothelium-Dependent Brachial Artery Vasoactivity Following a SingleHigh Fat Meal”, JAMA 278: 1682-1686 (Nov. 26, 1997), incorporated hereinby reference. According to the present invention, glutathione may beadministered as a prophylaxis against vascular disease.

In utilizing antioxidants as advanced therapeutic approaches, thefollowing principles have been developed over time:

Different disorders generate different types of free radicals, indifferent environments. Therefore, different specific antioxidants areneeded for these various radicals and related compounds. The commonestspecies and related molecules includes superoxide, .O₂—; hydroxyl, .OH;peroxy, .OOH; hydrogen peroxide, H₂O₂ (splitting into hydroxylradicals); alkoxy, RO.; delta singlet oxygen, ¹O₂; nitric oxide, .NO;lipid hydroperoxides, LOOH (splitting into alkoxy and hydroxylradicals). See, Montaignier, Luc, Olivier, Rene, Pasquier, Catherine(Eds.), Oxidative Stress in Cancer, AIDS, and NeurodegenerativeDiseases, Marcel Dekker, NY (1998), incorporated herein by reference inits entirety.

In addition to qualitative differences among several species of freeradicals, their rates of formation will differ, as will the differenttypes of inciting agents that may have to be simultaneously controlled.For example, continued, unprotected exposures of the eyes, in MacularDegeneration, to strong sunlight and to tobacco smoke, would limitbenefits from an antioxidant used as a therapeutic agent for control ofthis disease. Therefore, one aspect of the invention providessynergistic therapies to patients by increasing antioxidant levelssystemically or in specific organs as well as reducing oxidative, freeradical generating and ionizing influences. In this case, glutathionetherapy would be complemented with ultraviolet blocking sunglasses, anda tobacco smoking cessation plan, if necessary. A particularlyadvantageous antioxidant for combination with glutathione is alphatocopherol succinate.

Free radicals occur in different parts or subparts of tissues and cells,with different inciting agents. For example, in trauma to the brain orspinal cord, the injurious free radicals are in the fatty (lipid)coverings that insulate nerve fibers, the myelin sheaths. Extremely highdoses of a synthetic corticosteroid, 5 to 10 grams of methylprednisolone sodium succinate (MPSS), given for just 24 hours, rapidlyreach the brain and spinal cord and diffuse rapidly into the myelin,neutralizing the trauma-induced radicals, specifically: .OH, .OOH, andRO.. It is therefore an object of the invention to provide apharmaceutical composition comprising a combination of glutathione and aglucocorticoid agent.

TRX has been shown to modulate the signaling processes of programmedcell death (apoptosis). TRX and other thiol compounds exert a protectiveactivity against cytotoxicity and apoptosis induced by various oxidativestresses. For example, Fas and TNF-α dependent cell death may beprotected by intracellular as well as extracellular TRX. The activity ofthe ICE (interleukin 1b-converting enzyme) family proteases (caspases),with cysteine residue in their active site, which are involved inapoptosis, are regulated by a redox mechanism. For example, the activityof caspase-3 (CPP32), an important member of caspases, is markedlyinhibited by oxidizing agents, which is counteracted by dithiothreitolor TRX. In contrast, on exposure to diamide or hydogen peroxide, amarked increase of CPP32 protease activity was observed after a fewhours, suggesting that intracellular redox state profoundly modulatesthe signaling processes of apoptosis by regulating the activity ofcaspases. Many transcription factors and DNA-binding proteins are redoxregulated by TRX, including NF-κB, AP-1, PEBP2/AML-1, and p53. JunjiYodoil, Shugo Ueda, Masaya Ueno, Tetsuro Sasada, and Hiroshi Masutani,Redox control of Thioredoxin (TRX) on the cytotoxic/death signal.

Superoxide (O₂ ⁻) is the compound obtained when oxygen is reduced by oneelectron. Oxidants related to superoxide include H₂O₂ and alkylperoxides, hydroxyl radical and other reactive oxidizing radicals,oxidized halogens and halamines, singlet oxygen, and peroxynitrite.These molecules are thought to participate in the pathogenesis of anumber of common diseases, including among others malignancy, by theirability to mutate the genome, and atherosclerosis, by their capacity foroxidizing lipoproteins. Oxidizing agents are, however, arephysiologically important for host defense, where they serve asmicrobicidal and parasiticidal agents, in normal apoptosis, orprogrammed cell death, and in biological signaling, where theirliberation in small quantities results in redox-mediated changes in thefunctions of enzymes and other proteins. It is generally believed thathost defense mechanisms are mediated by such strong effects thatpharmacological antioxidants would not be able to overcome the powerfuloxidant effects. On the other hand, it is believed that antioxidantpharmaceuticals may play an important role in modulating redox-mediatedsignaling and early steps in biological cascades, such as apoptosis.

The accepted, published, peer-reviewed literature has repeatedlydemonstrated the multiple properties of glutathione in the body. Theabundant physiological and biochemical properties of glutathione ledothers into an extensive series of clinical trials wherein precursors ofglutathione were administered, because the prevailing belief was thatglutathione itself could not be effectively absorbed if it was simplygiven as glutathione. Hence, the popularity of the relativelyineffective and potentially damaging glutathione precursor N-acetylcysteine (NAC) is currently being misused in the homosexual (high AIDSrisk) community. The further belief was that glutathione would not crossthe membranes of lymphocytes and other cells, whereas NAC could. Theview was that to try to correct the glutathione deficiency in HIV/AIDS,with glutathione itself, was a hopeless task, because it would bedegraded before uptake across membranes. However, the precursors ofglutathione have failed to raise intracellular GSH levels. The presentinvention provides a suitable regimen to orally administer glutathioneto achieve high bioavailability and increased intracellular levels ofglutathione.

While prior studies have employed glutathione dissolved in orange juiceto administer glutathione to AIDS patients, resulting in glutathioneuptake, this method does not provide the advantages of an encapsulatedor pill form, and there was no recognition for the need to preventdigestive dilution or glutathione derived impurities from being present.

Glutathione has also proven to be an effective anti-viral agent andinterferes with HIV replication at a critical site that is not affectedby other current drugs, viral mRNA transcription. Glutathione keepsviral DNA quiescent, especially when potent activators are present, likeNF-κB, and TNF-α. Glutathione's anti-viral target appears to be at apoint where the virus can not readily mutate. The dependence of HIVreplication on binding activated NF-κB onto its Long Terminal Repeat(LTR) appears to be central to the virus.

According to the present invention, orally administered glutathione cansafely raise cell levels beyond correcting glutathione deficiencies. Anumber of pathologic processes can be inhibited by these higher levels,for example, curtailing the virtually self-perpetuating, powerfulbiochemical cycles producing corrosive free radicals and toxic cytokinesthat are largely responsible for the signs and symptoms of AIDS. Thesebiochemical cycles destroy considerable quantities of glutathione butthey can eventually be brought under control, and normalized withsufficient, on-going glutathione therapy. A typical example is the overproduction of a substance, 15 HPETE (15-hydroperoxy eicosatetraenoicacid), from activated macrophages. The 15 HPETE is a destructive,immunosuppressing substance and requires glutathione for conversion intoa non-destructive, benign molecule. The problem is that once macrophagesare activated, they're difficult to normalize.

Once inside cells, GSH curtails the production of free radicals andcytokines, corrects the dysfunctions of lymphocytes and of macrophages,reinforces defender cells in the lungs and other organs, halts HIVreplication in all major infected cell types, by preventing theactivation of the viral DNA by precluding the activation of NF-κB,inhibits the TAT gene product of HIV that drives viral replication,dismantles the gp120 proteins of the virus coat. These gp120 proteinsare the projections of the virus that normally allow it to lock ontosusceptible CD4+ cells thereby helping the spread of the virus touninfected CD4+ cells. By disrupting the gp120 protein, glutathioneoffers a potential mode of preventing transmission not only to othercells in the patient, but perhaps in precluding transmission to others.

Besides classic antiviral or antiretroviral agents (reversetranscriptase inhibitors, protease inhibitors), a number of othertherapies may be of benefit for AIDS patients, and the present inventionprovides combinations of glutathione with the following drugs:cyclosporin A, thalidomide, pentoxifylline, selenium, desferroxamine,2L-oxothiazolidine, 2L-oxothiazolidine-4-carboxylate,diethyldithiocarbamate (DDTC), BHA, nordihydroguairetic acid (NDGA),glucarate, EDTA, R-PIA, alpha-lipoic acid, quercetin, tannic acid,2′-hydroxychalcone, 2-hydroxychalcone, flavones, alpha-angelicalactone,fraxetin, curcurmin, probucol, and arcanut (areca catechul).

Inflammatory responses are accompanied by large oxidative bursts,resulting in large numbers of free radicals. Therefore, glutathione mayhave application in the therapy for inflammatory diseases. Glutathionemay advantageously reduce the primary insult a well as undesired aspectsof the secondary response. According to the present invention,glutathione may be administered to patients suffering from aninflammatory disease process, such as arthritis or various types,inflammatory bowel disease, etc. The present invention also providescombination pharmaceutical therapy including glutathione and ananalgesic or antiinflammatory agent, for example opiate agonists,glucocorticoids or non-steroidal antiinflammatory drugs (NSAIDS),including opium narcotics, meperidine, propoxyphene, nalbuphine,pentazocine, buprenorphine, asprin, indomethacin, diflunisal,acetominophen, ibuprofen, naproxen, fenoprofen, piroxicam, sulindac,tolmetin, meclofenamate, zomepirac, penicillamine, phenylbutazone,oxyphenbutazone, chloroquine, hydroxychloroquine, azathiaprine,cyclophosphamide, levamisole, prednisone, prednisolone, betamethasone,triamcinolone, and methylprednisolone.

Glutathione may also hold benefit for the treatment of parotitis,cervical dysplasia, Alzheimer's disease, Parkinson's disease,aminoquinoline toxicity, gentamycin toxicity, puromycin toxicity,aminoglycoside nephrotoxicity, paracetamol, acetaminophen and phenacetintoxicity.

Glutathione need not be orally ingested in order to provide thebeneficial effects noted. While the drug may be administeredintravenously or parenterally, it may also be administered throughmucous membranes, including sublingually, as a vaginal or rectalsuppository, and by pulmonary inhaler, for topical applications to thealveolar surface cells of the lungs to enhance pulmonary protectionagainst unusual pneumonias. Systemic administration of glutathione maybe used to concentrate glutathione in lymph nodes, and lymphoid tissues.

Glutathione tends to be unstable in solution. Therefore, one aspect ofthe present invention provides a pharmaceutical administration apparatusproviding a dual chamber distribution pouch, having a frangibleinterconnection, allowing mixing between an aqueous phase and a dryglutathione preparation. The aqueous phase may be, for example, a gel,cream or foam. Either pouch may also contain another pharmaceuticalagent, as described above.

The present invention also provides a glutathione administrationappliance, for delivering an effective dose of glutathione to anaccessible mucous membrane, such as the oral, vaginal, urethral or analcavities. A dry glutathione preparation, for example in a dehydratedgel, matrix or polymer, having a high surface area per unit volumeratio, is provided in a foil bag or pouch. The dehydrated mass includesglutathione, as well as an optional stabilizing agent, such as ascorbicacid. The dehydrated mass is hydrated by the mucosal membrane or by anexternally applied fluid, and the glutathione is then present to protectthe mucous membrane from viral infection.

The ability of glutathione to chemically dismantle the gp120 protein ofHIV by chemically destroying structural disulfide bonds, indicates thattransmission of the infection may be curtailed to some extent. If gp120is dismantled, the virus cannot lock onto CD4+ cells. The oralglutathione treatment of patients may suffice to dismantle gp120 ofviruses from treated patients. The topical applications of glutathioneto mucous membranes might possibly serve to protect a sex partner ifunsafe sexual practices occur.

Another effect is seen when glutathione or nitroso-glutathione is placedin the male urethra. In this case, the glutathione or glutathionederivative is absorbed. The vasodilatory effects of nitroso-glutathione,which is formed by interaction of glutathione with nitric oxide orprovided directly, vasodilates the penis, resulting in an erection.Thus, a urethral glutathione or nitroso-glutathione suppository haspotential for the treatment of impotence. Glutathione ornitroso-glutathione may also be used to treat female sexual dysfunction.Direct application of glutathione or nitroso-glutathione to the mucousmembranes, for example, as a cream or in a gel formulation, will resultin local vasodilation, lubrication, and engorgement of erectile tissue.

It is noted that the effects of various pharmacological agents which actto increase the production of nitric oxide, for example the substratefor formation of nitric oxide, the amino acid arginine, the stability ofnitric oxide in the blood, or the effect of nitric oxide, may be usedsynergistically. Likewise, drugs which act on differing systems, such asthe central nervous system and peripheral vascular system, may also beused synergistically. Thus, glutathione may be used alone or incombination to achieve its effects on the circulatory system andvascular tissues.

Glutathione or a glutathione derivative may also be co-administered withyohimbine, an alpha-2 receptor blocker, providing a synergistic effect.Yohimbine has been established to treat male sexual dysfunction, (e.g.,impotence), among other effects. Apomorphine may also providesynergistic effects with glutathione for the treatment of impotence. Itis noted that, in many cases, female sexual dysfunction may be relatedto pelvic and genital vascular response, in particular vasodilation, andtherefore glutathione, alone or in combination with other vasoactive orneuroactive substances, may be beneficial in the treatment of both maleand female sexual dysfunction.

Glutathione may be administered to mucous membranes in the form of aliquid, gel, cream, jelly, absorbed into a pad or sponge. Administrationmay also be provided by a powder or suspension.

The effective delivery of intact, pharmaceutically stabilized,bioavailable reduced L-glutathione has been accomplished according tothe present invention. By providing high-dose glutathione for the body'sgeneral use, diabetics having either form of the disease may be providedwith ample supplies of glutathione. Correcting the glutathionedeficiency and also raising the levels inside cells to the upper rangeof normal will help to delay, or prevent the complications of diabetes.

Glutathione, orally administered according to the present invention, inmoderately high doses, one to five gm/day, may be able to affect theoutcome of macular degeneration. The avidity with which the RPE cellstake up glutathione indicates that they may have a critical role inameliorating this disorder. Unlike rods and cones, RPE cells can divideand replenish themselves if allowed. If caught at an early stage, beforesignificant losses of rods and cones, the condition may be halted anddelayed possibly indefinitely.

Since glutathione is relatively non-toxic, it may be used liberally forits advantageous properties. According to one aspect of the invention,glutathione may be added to a viral contaminated fluid or potentiallycontaminated fluid to inactivate the virus. This occurs, for example, byreduction of critical viral proteins. According to a preferredembodiment, glutathione is added to blood or blood components prior totransfusion. The added glutathione is in the reduced form, and is addedin a concentration of between about 100 micromolar to about 500millimolar to a solubility limit, whichever is lower, and morepreferably in a concentration of about 10-50 millimolar.

The addition of glutathione to whole blood, packed red blood cells orother formed blood components (white blood cells, platelets) may be usedto increase the shelf life and/or quality of the cells or formedcomponents.

It is also noted that other pharmacological agents may be employed toachieve alterations in redox balance or to acts free radical scavengingagents. These may be employed individually or in combination. Forexample, glutathione may also be administered in conjunction with otherantioxidants or redox-active drugs; a preferred formulation for oraladministration of glutathione according to the present inventionincludes ascorbic acid (Vitamin C). Other acceptable agents foradministration include α-tocopherol, either in the free state as anantioxidant or as a pharmaceutically acceptable ester thereof as aVitamin E precursor. In addition, α lipoic acid is believed to be anontoxic, orally bioavailable and effective antioxidant. It is noted,however, that glutathione is a most preferred agent due to its centralrole in maintaining cell oxidative balance, ubiquity in the body, andhigh therapeutic index. According to the present invention, onetraditional difficulty, obtaining high oral bioavailability forglutathione, has been solved.

EXAMPLE 1

Reduced L-glutathione, a naturally-occurring water-soluble tripeptide(gamma-glutamyl-cysteinyl-glycine) is the most prevalent intracellularthiol in most biological systems. A preferred formulation of glutathioneaccording to the present invention provides capsules for oral usecontaining 500 mg reduced L-glutathione, 250 mg USP grade crystallineascorbic acid, and not more than 0.9 mg magnesium stearate, NF grade inan OO-type gelatin capsule.

EXAMPLE 2

The preferred regimen for treatment of humans with glutathione accordingto the present invention is the administration of between 1 and threegrams per day, in two divided doses, between meals (on an emptystomach), of encapsulated, stabilized glutathione according toExample 1. The study detailed in Appendix B administered the glutathioneto HIV infected, otherwise healthy males between 18 and 65, with CD4+cell counts above 500, not on any other medications. As detailed in FIG.1, clinical responses were seen in the PBM intracellular glutathionelevels. Thus, at 1 hour after administration of a 1-gram bolus ofencapsulated stabilized glutathione in two 500 mg capsules, a three-foldincrease in glutathione was measured. It is noted that, since the humanbody produces large quantities of glutathione, the effects of externalglutathione in individual cases may sometimes be masked or even appearparadoxical. However, as shown in FIG. 2, a statistical analysis shows adose response effect of the administration of glutathione according tothe present invention to the subject population.

EXAMPLE 3

FIG. 2 shows a graph from one of the 24 HIV positive people in theCompany's Clinical Trial. The graph illustrates increases in theglutathione (GSH) content of immune system cells, in the blood,resulting from two doses of pharmaceutically stabilized GSH according toExample 1. The first dose of one gram was taken at 0 time, or 10:00a.m., and the second dose at 3 hours, or 1:00 p.m. The baseline pointswere from two weeks earlier, on the same patient. A temporaryintravenous catheter was in place for 7 hours to permit frequent bloodsampling at the numerous time points. The units are in nanomoles of GSHper 10 million peripheral blood mononuclear cells (PBMC's). The graph isan example of the elevation of GSH inside PBMC's. The statisticalanalysis of the entire patient population shows statisticallysignificant elevations and a significant dose response relationship.

In a compressed Phase I/II clinical trial (FDA IND#45012), in awell-defined GSH deficiency state, HIV infection, the compositionaccording to Example 1 administered according to the protocol of Example2 was demonstrated to rapidly and safely raises intracellular GSH levelstwo to three fold. Thus, by employing the composition according toExample 1 administered according to the protocol of Example 2, an oralpharmaceutical has been shown to treat the critical losses of GSH thatare known to propel a range of major disorders.

The glutathione metabolism, especially the pharmacokinetics, of thesubjects of the Phase II study is believed to be relatively normal.Therefore, the same regimen may be applied in the treatment of otherconditions, including CHF, diabetes, early stroke or other ischemicevent, toxic insult, viral infection or disease, or other condition inwhich free radical reactions are uncontrolled, aberrant, or contributeto pathology.

EXAMPLE 4 Combination of Glutathione and Acetaminophen

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of acetaminophen, a drug which consumes glutathione in the liverduring metabolism, and in excess doses causes liver damage due tooxidative damage. The composition includes 500 mg L-glutathione, 250 mgcrystalline ascorbic acid, and 350 mg acetaminophen.

EXAMPLE 5 Combination of Glutathione and Chlorpromazine

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of chlorpromazine, a phenothiazine drug that causes sideeffects, including tardive dyskinesia, possibly relating to excess freeradical reactions. The composition includes 500 mg L-glutathione, 250 mgcrystalline ascorbic acid, and 200 mg chlorpromazine.

EXAMPLE 6 Combination of Glutathione and Aminoglycosides

A combination pharmaceutical is provided to ameliorate the detrimentaleffects of Aminoglycoside drugs, which include, but are not limited to,neomycin, kanamycin, amikacin, streptomycin, gentamycin, sisomicin,netilmicin and tobramycin, a drug class which may be associated withvarious toxicities. This damage may be related to oxidative damage orconsumption of glutathione during metabolism. The composition accordingto the present invention is an intravenous formulation, including theaminoglycoside in an effective amount, and L-glutathione in an amount ofabout 10-20 mg/kg. Ascorbic acid in an amount of 5-10 mg/kg may be addedas a stabilizer.

EXAMPLE 7 Urethral Insert

A composition containing 200 mg glutathione, 50 mg ascorbic acid perunit dosage is mixed with carageenan and/or agarose and water in aquick-gelling composition, and permitted to gel in a cylindrical formhaving a diameter of about 3 mm and a length of about 30 mm. Thecomposition is then subjected to nitric oxide to cause between 0.1-10%of the glutathione to be converted to nitroso-glutathione. The gelledagarose is then freeze-dried under conditions which allow shrinkage. Thefreeze-dried gel is than packaged in a gas barrier package, such as afoil pouch or foil “bubble-pack”.

The freeze-dried gel may then be used as a source of nitroso-glutathionefor administration transmucosally. The cylindrical freeze-dried gel maybe inserted into the male urethra for treatment of impotence, oradministered sublingually for systemic vasodilation.

EXAMPLE 7 Vascular Disease Prophylaxis

An oral formulation is provided for prophylaxis of vascular disease,e.g., in men over 40. The composition includes 500 mg reducedL-glutathione, 250 mg USP grade crystalline ascorbic acid, and 50 mg USPacetyl salicylic acid (aspirin) in an OO-type gelatin capsule. Typicaladministration is twice per day.

Advantageously, the acetyl salicylic acid may provided in entericrelease pellets within the capsule, slowing release.

EXAMPLE 8 Vascular Disease Prophylaxis

Arginine is the normal starting substrate for the production of nitricoxide. Arginine is normally in limited supply, and thus a relativedeficiency of arginine may result in impaired vascular endothelialfunction.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 200 mg arginine, in an OO-type gelatincapsule.

EXAMPLE 9 Vascular Disease Prophylaxis

Vitamin E consumption reduces the risk of heart attack and othervascular disease. Vitamin E succinate (alpha-tocopherol succinate) is adry powder.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 200 mg vitamin E succinate, in an OO-typegelatin capsule.

EXAMPLE 10 Vascular Disease Prophylaxis

Nonspecific esterases are present in the plasma that have a broadsubstrate specificity. According to the present invention, esters areformed between agents that are useful combination therapies, in order toprovide for efficient administration, high bioavailability, andpharmaceutical stability. Preferred esters include alphatocopherol-ascorbate, alpha tocopherol-salicylate, andascorbyl-salicylate. The tocopherol ester maintains the molecule in areduced state, allowing full antioxidant potential after ester cleavage.

These esters may be administered alone or in combination with otheragents, for example glutathione. Typically, these are administered todeliver an effective dose of salicylate equivalent of 100 mg per day forprophylaxis or 750-1000 mg per dose for treatment of inflammatorydiseases. Tocopherol is administered in an amount of 100-500 IUequivalent. Ascorbate is administered in an amount of up to 1000 mgequivalent.

In order to enhance availability, a non-specific esterase may beprovided in the formulation to cleave the ester after dissolution of thecapsule. Therefore, a non-specific esterase, such as a bacterial orsaccharomyces (yeast) enzyme or enriched enzyme preparation may beincluded in the formulation, such as included as a powder or as pelletsin the capsule.

EXAMPLE 11 Vascular Disease Prophylaxis

Nordihydroguaretic acid is a known lipoxygenase inhibitor. Thiscomposition may therefore be used to treat inflammatory processes or asprophylaxis against vascular disease.

An oral formulation is provided for prophylaxis of vascular disease. Thecomposition includes 500 mg reduced L-glutathione, 200 mg USP gradecrystalline ascorbic acid, and 100 mg nordihydroguaretic acid, in anOO-type gelatin capsule. Typical administration is twice per day.

The references and patents hereinabove recited are expresslyincorporated herein by reference.

It should be understood that the preferred embodiments and examplesdescribed herein are for illustrative purposes only and are not to beconstrued as limiting the scope of the present invention, which isproperly delineated only in the appended claims.

What is claimed is:
 1. A method, comprising administering a sufficientoral dose of reduced glutathione to alter a cellular redox potential andthereby modify production of gene products.
 2. The method according toclaim 1, wherein the gene product id PEDF.
 3. The method according toclaim 1, wherein the gene product is a viral protein.
 4. The methodaccording to claim 1, wherein the gene product is a paracrine growthhormone.
 5. The method according to claim 1, wherein the gene product isup-regulated by a reductive shift in redox potential.
 6. The methodaccording to claim 1, wherein the gene product is down-regulated by areductive shift in redox potential.
 7. The method according to claim 1wherein the glutathione is formulated as encapsulated pharmaceuticallystabilized glutathione in a rapidly dissolving formulation comprisesabout 500 mg of glutathione and about 250 mg of crystalline ascorbicacid in a hard gelatin capsule.
 8. The method according to claim 1,wherein the glutathione is pharmaceutically stabilized with ascorbicacid.
 9. The method according to claim 8, wherein the ascorbic acid ispresent in an amount of about 1:1 to 1:10 to glutathione by weight. 10.The method according to claim 1 wherein the glutathione is encapsulatedwith an antistatic agent.
 11. The method according to claim 10, whereinthe antistatic agent is crystalline ascorbic acid.
 12. The methodaccording to claim 1, wherein the glutathione prevents growth ofneoplasms.
 13. The method according to claim 1, wherein the glutathioneis administered as a bolus on an empty stomach.
 14. A method of alteringan expression of gene products in mammalian cells comprising orallyadministering reduced glutathione to achieve an effective concentrationin the duodenum of at least about 500 micromolar, with less than about10 grams of food present per gram of glutathione in the duodenum.